Antibody composition-producing cell

ABSTRACT

The present invention relates to a cell for the production of an antibody molecule such as an antibody useful for various diseases having high antibody-dependent cell-mediated cytotoxic activity, a fragment of the antibody and a fusion protein having the Fc region of the antibody or the like, a method for producing an antibody composition using the cell, the antibody composition and use thereof.

This is a divisional of application Ser. No. 13/102,565, filed May 6,2011 (U.S. Pat. No. 8,329,443), which is a continuation of applicationSer. No. 12/628,083, filed Nov. 30, 2009 (abandoned), which is acontinuation of application Ser. No. 11/218,473, filed Sep. 6, 2005 (nowU.S. Pat. No. 7,741,442), which is a divisional of application Ser. No.09/971,773 filed Oct. 9, 2001 (now U.S. Pat. No. 6,946,292), whichclaims benefit of Provisional Application No. 60/268,916 filed Feb. 16,2001, International Application No. PCT/JP01/08804 filed Oct. 5, 2001,and Japanese Patent Application No. 2000-308526 filed Oct. 6, 2000. Theentire disclosures of the prior applications are considered part of thedisclosure and are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a cell for the production of anantibody molecule such as an antibody useful for various diseases, afragment of the antibody and a fusion protein having the Fc region ofthe antibody or the like, a method for producing an antibody compositionusing the cell, the antibody composition and use thereof.

2. Brief Description of the Background Art

Since antibodies have high binding activity, binding specificity andhigh stability in blood, their applications to diagnosis, prevention andtreatment of various human diseases have been attempted [MonoclonalAntibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 2.1(1995)]. Also, production of a humanized antibody such as a humanchimeric antibody or a human complementarity determining region(hereinafter referred to as “CDR”)-grafted antibody from an antibodyderived from an animal other than human have been attempted by usinggenetic recombination techniques. The human chimeric antibody is anantibody in which its antibody variable region (hereinafter referred toas “V region”) is an antibody derived from an animal other than humanand its constant region (hereinafter referred to as “C region”) isderived from a human antibody. The human CDR-grafted antibody is anantibody in which the CDR of a human antibody is replaced by CDR of anantibody derived from an animal other than human.

It has been revealed that five classes, namely IgM, IgD, IgG, IgA andIgE, are present in antibodies derived from mammals. Antibodies of humanIgG class are mainly used for the diagnosis, prevention and treatment ofvarious human diseases because they have functional characteristics suchas long half-life in blood, various effector functions and the like[Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc.,Chapter 1 (1995)]. The human IgG class antibody is further classifiedinto the following 4 subclasses: IgG1, IgG2, IgG3 and IgG4. A largenumber of studies have so far been conducted for antibody-dependentcell-mediated cytotoxic activity (hereinafter referred to as “ADCCactivity”) and complement-dependent cytotoxic activity (hereinafterreferred to as “CDC activity”) as effector functions of the IgG classantibody, and it has been reported that among antibodies of the humanIgG class, the IgG1 subclass has the highest ADCC activity and CDCactivity [Chemical Immunology, 65, 88 (1997)]. In view of the above,most of the anti-tumor humanized antibodies, including commerciallyavailable Rituxan and Herceptin, which require high effector functionsfor the expression of their effects, are antibodies of the human IgG1subclass.

Expression of ADCC activity and CDC activity of the human IgG1 subclassantibodies requires binding of the Fc region of the antibody to anantibody receptor existing on the surface of an effector cell, such as akiller cell, a natural killer cell, an activated macrophage or the like(hereinafter referred to as “FcγR”) and various complement componentsare bound. Regarding the binding, it has been suggested that severalamino acid residues in the hinge region and the second domain of Cregion (hereinafter referred to as “Cγ2 domain”) of the antibody areimportant [Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319(1995), Chemical Immunology, 65, 88 (1997)] and that a sugar chainbinding to the Cγ2 domain [Chemical Immunology, 65, 88 (1997)] is alsoimportant.

Regarding the sugar chain, Boyd et al. have examined effects of a sugarchain on the ADCC activity and CDC activity by treating a humanCDR-grafted antibody CAMPATH-1H (human IgG1 subclass) produced by aChinese hamster ovary cell (CHO cell) or a mouse myeloma NSO cell (NSOcell) with various sugar hydrolyzing enzymes, and reported thatelimination of the non-reducing end sialic acid did not have influenceupon both activities, but the CDC activity alone was affected by furtherremoval of galactose residue and about 50% of the activity wasdecreased, and that complete removal of the sugar chain causeddisappearance of both activities [Molecular Immunol., 32, 1311 (1995)].Also, Lifely et al. have analyzed the sugar chain bound to a humanCDR-grafted antibody CAMPATH-1H (human IgG1 subclass) which was producedby CHO cell, NSO cell or rat myeloma YO cell, measured its ADCCactivity, and reported that the CAMPATH-1H derived from YO cell showedthe highest ADCC activity, suggesting that N-acetylglucosamine(hereinafter referred also to as “GlcNAc”) at the bisecting position isimportant for the activity [Glycobiology, 5, 813 (1995); WO 99/54342].These reports indicate that the structure of the sugar chain plays animportant role in the effector functions of human antibodies of IgG1subclass and that it is possible to prepare an antibody having morehigher effector function by changing the structure of the sugar chain.However, actually, structures of sugar chains are various and complex,and it cannot be said that an actual important structure for theeffector function was identified.

Sugar chains of glycoproteins are roughly divided into two types, namelya sugar chain which binds to asparagine (N-glycoside-linked sugar chain)and a sugar chain which binds to other amino acid such as serine,threonine (O-glycoside-linked sugar chain), based on the binding form tothe protein moiety. The N-glycoside-linked sugar chains have variousstructures [Biochemical Experimentation Method 23—Method for StudyingGlycoprotein Sugar Chain (Gakujutsu Shuppan Center), edited by ReikoTakahashi (1989)], but it is known that they have a basic common corestructure shown by the following structural formula (I).

The sugar chain terminus which binds to asparagine is called a reducingend, and the opposite side is called a non-reducing end. It is knownthat the N-glycoside-linked sugar chain includes a high mannose type inwhich mannose alone binds to the non-reducing end of the core structure;a complex type in which the non-reducing end side of the core structurehas at least one parallel branches of galactose-N-acetylglucosamine(hereinafter referred to as “Gal-GlcNAc”) and the non-reducing end sideof Gal-GlcNAc has a structure of sialic acid, bisectingN-acetylglucosamine or the like; a hybrid type in which the non-reducingend side of the core structure has branches of both of the high mannosetype and complex type; and the like.

In the Fc region of an antibody of an IgG type, two N-glycoside-linkedsugar chain binding sites are present. In serum IgG, to the sugar chainbinding site, generally, binds a complex type sugar chain having pluralbranches and in which addition of sialic acid or bisectingN-acetylglucosamine is low. It is known that there is variety in themanner and form in which addition of galactose is made to thenon-reducing end of the complex type sugar chain and the addition offucose to the N-acetylglucosamine in the reducing end [Leppanen, A, etal., Biochemistry, (1997) 36, 7026-7036, “in vitro Biosynthesis of aDecasacharide Prototype of Multiply Branched PolylactoaminoglycanBackbones”].

It has been considered that such a structure of a sugar chain isdetermined by sugar chain genes, namely a gene for a glycosyltransferasewhich synthesizes a sugar chain and a gene for a glycolytic enzyme whichhydrolyzes the sugar chain.

Synthesis of an N-glycoside-linked sugar chain is described below.

Glycoproteins are modified with a sugar chain in the endoplasmicreticulum (hereinafter referred to as “ER”) lumen. During thebiosynthesis step of the N-glycoside-linked sugar chain, a relativelylarge sugar chain is transferred to the polypeptide chain which iselongating in the ER lumen. In the transformation, the sugar chain isfirstly added in succession to phosphate groups of a long chain lipidcarrier comprising about 20 α-isoprene units, which is called dolicholphosphate (hereinafter referred also to as “P-Dol”). That is,N-acetylglucosamine is transferred to dolichol phosphate to thereby formGlcNAc-P-P-Dol and then one more GlcNAc is transferred to formGlcNAc-GlcNAc-P-P-Dol. Next, five mannoses (hereinafter mannose is alsoreferred to as “Man”) are transferred to thereby form(Man)₅-(GlcNAc)₂-P-P-Dol and then four Man's and three glucoses(hereinafter glucose is also referred to as “Glc”) are transferred.Thus, a sugar chain precursor, (Glc)₃-(Man)₉-(GlcNAc)₂-P-P-Dol, calledcore oligosaccharide is formed. The sugar chain precursor comprising 14sugars is transferred as a mass to a polypeptide having anasparagine-X-serine or asparagine-X-threonine sequence in the ER lumen.In the reaction, dolichol pyrophosphate (P-P-Dol) bound to the coreoligosaccharide is released but again becomes dolichol phosphate byhydrolysis with pyrophosphatase and is recycled. Trimming of the sugarchain immediately starts after the sugar chain binds to the polypeptide.That is, 3 Glc's and 1 or 2 Man's are eliminated on the ER, and it isknown that α-1,2-glucosidase I, α-1,3-glucosidase II andα-1,2-mannosidase relates to the elimination.

The glycoprotein which was subjected to trimming on the ER istransferred to the Golgi body and are variously modified. In the cispart of the Golgi body, N-acetylglucosamine phosphotransferase whichrelates to addition of mannose phosphate, N-acetylglucosamine1-phosphodiester α-N-acetylglucosaminidase and α-mannosidase I arepresent and reduce the Man residues to 5. In the medium part of theGolgi body, N-acetylglucosamine transferase I (GnTI) which relates toaddition of the first outside GlcNAc of the complex typeN-glycoside-linked sugar chain, α-mannosidase II which relates toelimination of 2 Man's, N-acetylglucosamine transferase II (GnIII) whichrelates to addition of the second GlcNAc from the outside andα-1,6-fucosyltransferase which relates to addition of fucose to thereducing end N-acetylglucosamine are present. In the trans part of theGolgi body, galactose transferase which relates to addition of galactoseand sialyltransferase which relates to addition of sialic acid such asN-acetylneuraminic acid or the like are present. It is known thatN-glycoside-linked sugar chain is formed by activities of these variousenzymes.

In general, most of the humanized antibodies of which application tomedicaments is in consideration are prepared using genetic recombinationtechniques and produced using Chinese hamster ovary tissue-derived CHOcell as the host cell. But as described above, since the sugar chainstructure plays a remarkably important role in the effector function ofantibodies and differences are observed in the sugar chain structure ofglycoproteins expressed by host cells, development of a host cell whichcan be used for the production of an antibody having higher effectorfunction is desired.

In order to modify the sugar chain structure of the producedglycoprotein, various methods have been attempted, such as 1)application of an inhibitor against an enzyme relating to themodification of a sugar chain, 2) selection of a mutant, 3) introductionof a gene encoding an enzyme relating to the modification of a sugarchain, and the like. Specific examples are described below.

Examples of an inhibitor against an enzyme relating to the modificationof a sugar chain include tunicamycin which selectively inhibitsformation of GlcNAc-P-P-Dol which is the first step of the formation ofa core oligosaccharide which is a precursor of an N-glycoside-linkedsugar chain, castanospermin and N-methyl-1-deoxynojirimycin which areinhibitors of glycosidase I, bromocondulitol which is an inhibitor ofglycosidase II, 1-deoxynojirimycin and 1,4-dioxy-1,4-imino-D-mannitolwhich are inhibitors of mannosidase I, swainsonine which is an inhibitorof mannosidase II and the like. Examples of an inhibitor specific for aglycosyltransferase include deoxy derivatives of substrates againstN-acetylglucosamine transferase V (GnTV) and the like [GlycobiologySeries 2—Destiny of Sugar Chain in Cell (Kodan-sha Scientific), editedby Katsutaka Nagai, Senichiro Hakomori and Akira Kobata (1993)]. Also,it is known that 1-deoxynojirimycin inhibits synthesis of a complex typesugar chain and increases the ratio of high mannose type and hybrid typesugar chains. Actually, it has been reported that sugar chain structureof IgG was changed and properties such as antigen binding activity andthe like was changed when the inhibitors were added to a medium[Molecular Immunol., 26, 1113 (1989)].

Mutants regarding the activity of an enzyme relating to the modificationof a sugar chain are mainly selected and obtained as a lectin-resistantcell line. For example, CHO cell mutants having various sugar chainstructures have been obtained as a lectin-resistant cell line using alectin such as WGA (wheat-germ agglutinin derived from T. vulgaris),ConA (cocanavalin A derived from C. ensiformis), RIC (a toxin derivedfrom R. communis), L-PHA (leucoagglutinin derived from P. vulgaris), LCA(lentil agglutinin derived from L. culinaris), PSA (pea lectin derivedfrom P. sativum) or the like [Somatic Cell Mol. Genet., 12, 51 (1986)].

As an example of the modification of the sugar chain structure of aproduct obtained by introducing the gene of an enzyme relating to themodification of a sugar chain into a host cell, it has been reportedthat a protein in which a number of sialic acid is added to thenon-reducing end of the sugar chain can be produced by introducing ratβ-galactoside-α-2,6-sialyltransferase into CHO cell [J. Biol. Chem.,261, 13848 (1989)].

Also, it was confirmed that an H antigen (Fuca-1-2Galβ1-) in whichfucose (hereinafter also referred to as “Fuc”) was added to thenon-reducing end of the sugar chain was expressed by introducing humanβ-galactoside-2-α-fucosyltransferase into mouse L cell [Science, 252,1668 (1991)]. In addition, based on knowledge that addition of thebisecting-positioned N-acetylglucosamine of N-glycoside-linked sugarchain is important for the ADCC activity of antibody, Umana et al. haveprepared CHO cell which expresses β-1,4-N-acetylglucosamine transferaseIII (GnTIII) and compared it with the parent cell line on the expressionof GnTIII. It was confirmed that express of GnTIII was not observed inthe parent cell line of CHO cell [J. Biol. Chem., 261, 13370 (1984)],and that the antibody expressed using the produced GnTIII expressing CHOcell had ADCC activity 16 times higher than the antibody expressed usingthe parent cell line [Glycobiology, 5, 813 (1995): WO 99/54342]. At thistime, Umana et al. have also produced CHO cell into whichβ-1,4-N-acetylglucosamine transferase V (GnTV) was introduced andreported that excess expression of GnTIII or GnTV shows toxicity for CHOcell.

Thus, in order to modify the sugar chain structure of the glycoproteinto be produced, control of the activity of the enzyme relating to themodification of a sugar chain in the host cell has been attempted, butactually, the structures of sugar chains are various and complex, andsolution of the physiological roles of sugar chains would beinsufficient, so that trial and error are repeated. Particularly,although it has been revealed little by little that the effectorfunction of antibodies is greatly influenced by the sugar chainstructure, a truly important sugar chain structure has not beenspecified yet. Accordingly, identification of a sugar chain which hasinfluence upon the effector function of antibodies and development of ahost cell to which such a sugar chain structure can be added areexpected for developing medicaments.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a host cell whichproduces an antibody composition and can control a sugar chain structurebound to an antibody molecule, a cell which can produce an antibodycomposition having high ADCC activity, a production method of anantibody composition using the cell and an antibody composition producedby the production method.

The present invention relates to the following (1) to (61).

(1) A Chinese hamster ovary tissue-derived CHO cell into which a geneencoding an antibody molecule is introduced, which produces an antibodycomposition comprising an antibody molecule having complexN-glycoside-linked sugar chains bound to the Fc region, wherein amongthe total complex N-glycoside-linked sugar chains bound to the Fc regionin the composition, the ratio of a sugar chain in which fucose is notbound to N-acetylglucosamine in the reducing end in the sugar chain is20% or more.(2) The CHO cell according to (1), wherein the sugar chain to whichfucose is not bound is a complex N-glycoside-linked sugar chain in which1-position of fucose is not bound to 6-position of N-acetylglucosaminein the reducing end through α-bond.(3) The CHO cell according to (1) or (2), wherein the antibody moleculebelongs to an IgG class.(4) The CHO cell according to any one of (1) to (3), wherein theactivity of an enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose and/or the activity of an enzyme relatingto the modification of a sugar chain in which 1-position of fucose isbound to 6-position of N-acetylglucosamine in the reducing end throughα-bond in the complex N-glycoside-linked sugar chain is decreased ordeleted.(5) The CHO cell according to (4), wherein the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose is an enzymeselected from the group consisting of the following (a), (b) and (c):

(a) GMD (GDP-mannose 4,6-dehydratase);

(b) Fx (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase);

(c) GFPP (GDP-beta-L-fucose pyrophosphorylase).

(6) The CHO cell according to (5), wherein the GMD is a protein encodedby a DNA of the following (a) or (b):

(a) a DNA comprising the nucleotide sequence represented by SEQ IDNO:65;

(b) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:65 under stringent conditions andencodes a protein having GMD activity.

(7) The CHO cell according to (5), wherein the GMD is a protein selectedfrom the group consisting of the following (a), (b) and (c):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:71;

(b) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:71 and has GMD activity;

(c) a protein which comprises an amino acid sequence having a homologyof at least 80% with the amino acid sequence represented by SEQ ID NO:71and has GMD activity.

(8) The CHO cell according to (5), wherein the Fx is a protein encodedby a DNA of the following (a) or (b):

(a) a DNA comprising the nucleotide sequence represented by SEQ IDNO:48;

(b) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:48 under stringent conditions andencodes a protein having Fx activity.

(9) The CHO cell according to (5), wherein the Fx is a protein selectedfrom the group consisting of the following (a), (b) and (c):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:72;

(b) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:72 and has Fx activity;

(c) a protein which comprises an amino acid sequence having a homologyof at least 80% with the amino acid sequence represented by SEQ ID NO:72and has Fx activity.

(10) The CHO cell according to (5), wherein the GFPP is a proteinencoded by a DNA of the following (a) or (b):

(a) a DNA comprising the nucleotide sequence represented by SEQ IDNO:51;

(b) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:51 under stringent conditions andencodes a protein having GFPP activity.

(11) The CHO cell according to (5), wherein the GFPP is a proteinselected from the group consisting of the following (a), (b) and (c):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:73;

(b) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:73 and has GFPP activity;

(c) a protein which comprises an amino acid sequence having a homologyof at least 80% with the amino acid sequence represented by SEQ ID NO:73and has GFPP activity.

(12) The CHO cell according to (4), wherein the enzyme relating to themodification of a sugar chain in which 1-position of fucose is bound to6-position of the N-acetylglucosamine in the reducing end through α-bondin the complex N-glycoside-linked sugar chain isα-1,6-fucosyltransferase.(13) The CHO cell according to (12), wherein theα-1,6-fucosyltransferase is a protein encoded by a DNA of the following(a) or (b):

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1;

(b) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:1 under stringent conditions andencodes a protein having α-1,6-fucosyltransferase activity.

(14) The CHO cell according to (12), wherein theα-1,6-fucosyltransferase is a protein selected from the group consistingof the following (a), (b) and (c):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:23;

(b) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:23 and hasα-1,6-fucosyltransferase activity;

(c) a protein which comprises an amino acid sequence having a homologyof at least 80% with the amino acid sequence represented by SEQ ID NO:23and has α-1,6-fucosyltransferase activity.

(15) The CHO cell according to any one of (4) to (14),

wherein the enzyme activity is decreased or deleted by a techniqueselected from the group consisting of the following (a), (b), (c), (d)and (e):

(a) a gene disruption technique targeting a gene encoding the enzyme;

(b) a technique for introducing a dominant negative mutant of a geneencoding the enzyme;

(c) a technique for introducing mutation into the enzyme;

(d) a technique for inhibiting transcription or translation of a geneencoding the enzyme;

(e) a technique for selecting a cell line resistant to a lectin whichrecognizes a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex N-glycoside-linked sugar chain.

(16) The CHO cell according to any one of (4) to (15), which isresistant to at least a lectin which recognizes a sugar chain in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the complex N-glycoside-linked sugarchain.(17) The CHO cell according to any one of (4) to (16), which produces anantibody composition having higher antibody-dependent cell-mediatedcytotoxic activity than an antibody composition produced by its parentCHO cell.(18) The CHO cell according to (17), which produces an antibodycomposition having higher antibody-dependent cell-mediated cytotoxicactivity than an antibody composition in which among the total complexN-glycoside-linked sugar chains bound to the Fc region contained in theantibody composition, the ratio of a sugar chain in which fucose is notbound to N-acetylglucosamine in the reducing end in the sugar chain isless than 20%.(19) The CHO cell according to (18), wherein the sugar chain to whichfucose is not bound is a complex N-glycoside-linked sugar chain in which1-position of fucose is not bound to 6-position of N-acetylglucosaminein the reducing end through α-bond.(20) A method for producing an antibody composition, which comprisesculturing the CHO cell according to any one of (1) to (19) in a mediumto produce and accumulate an antibody composition in the culture; andrecovering the antibody composition from the culture.(21) An antibody composition which is produced using the methodaccording to (20).(22) An antibody composition which comprises an antibody molecule havingcomplex N-glycoside-linked sugar chains bound to the Fc region which isproduced by a CHO cell, wherein among the total complexN-glycoside-linked sugar chains bound to the Fc region in thecomposition, the ratio of a sugar chain in which fucose is not bound toN-acetylglucosamine in the reducing end in the sugar chain is 20% ormore.(23) A cell in which the activity of an enzyme relating to the synthesisof an intracellular sugar nucleotide, GDP-fucose and/or the activity ofan enzyme relating to the modification of a sugar chain wherein1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the complex N-glycoside-linked sugarchain is decreased or deleted by a genetic engineering technique.(24) The cell according to (23), wherein the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose is an enzymeselected from the group consisting of the following (a), (b) and (c):

(a) GMD (GDP-mannose 4,6-dehydratase);

(b) Fx (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase);

(c) GFPP (GDP-beta-L-fucose pyrophosphorylase).

(25) The cell according to (24), wherein the GMD is a protein encoded bya DNA of the following (a) or (b):

(a) a DNA comprising the nucleotide sequence represented by SEQ IDNO:65;

(b) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:65 under stringent conditions andencodes a protein having GMD activity.

(26) The cell according to (24), wherein the GMD is a protein selectedfrom the group consisting of the following (a), (b) and (c):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:71;

(b) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:71 and has GMD activity;

(c) a protein which comprises an amino acid sequence having a homologyof at least 80% with the amino acid sequence represented by SEQ ID NO:71and has GMD activity.

(27) The cell according to (24), wherein the Fx is a protein encoded bya DNA of the following (a) or (b):

(a) a DNA comprising the nucleotide sequence represented by SEQ IDNO:48;

(b) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:48 under stringent conditions andencodes a protein having Fx activity.

(28) The cell according to (24), wherein the Fx is a protein selectedfrom the group consisting of the following (a), (b) and (c):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:72;

(b) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:72 and has Fx activity;

(c) a protein which comprises an amino acid sequence having a homologyof at least 80% with the amino acid sequence represented by SEQ ID NO:72and has Fx activity.

(29) The cell according to (24), wherein the GFPP is a protein encodedby a DNA of the following (a) or (b):

(a) a DNA comprising the nucleotide sequence represented by SEQ IDNO:51;

(b) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:51 under stringent conditions andencodes a protein having GFPP activity.

(30) The cell according to (24), wherein the GFPP is a protein selectedfrom the group consisting of the following (a), (b) and (c):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:73;

(b) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:73 and has GFPP activity;

(c) a protein which comprises an amino acid sequence having a homologyof at least 80% with the amino acid sequence represented by SEQ ID NO:73and has GFPP activity.

(31) The cell according to (23), wherein the enzyme relating to themodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe N-glycoside-linked sugar chain is α-1,6-fucosyltransferase.(32) The cell according to (31), wherein the α-1,6-fucosyltransferase isa protein encoded by a DNA selected from the group consisting of thefollowing (a), (b), (c) and (d):

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1;

(b) a DNA comprising the nucleotide sequence represented by SEQ ID NO:2;

(c) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:1 under stringent conditions andencodes a protein having α-1,6-fucosyltransferase activity;

(d) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:2 under stringent conditions andencodes a protein having α-1,6-fucosyltransferase activity.

(33) The cell according to (31), wherein the α-1,6-fucosyltransferase isa protein selected from the group consisting of the following (a), (b),(c), (d), (e) and (f):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:23;

(b) a protein comprising the amino acid sequence represented by SEQ IDNO:24;

(c) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:23 and hasα-1,6-fucosyltransferase activity;

(d) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:24 and hasα-1,6-fucosyltransferase activity;

(e) a protein which comprises an amino acid sequence having a homologyof at least 80% with the amino acid sequence represented by SEQ ID NO:23and has α-1,6-fucosyltransferase activity;

(f) a protein which comprises an amino acid sequence having a homologyof at least 80% with the amino acid sequence represented by SEQ ID NO:24and has α-1,6-fucosyltransferase activity.

(34) The cell according to any one of (23) to (33), wherein the geneticengineering technique is a technique selected from the group consistingof the following (a), (b), (c) and (d):

(a) a gene disruption technique targeting a gene encoding the enzyme;

(b) a technique for introducing a dominant negative mutant of a geneencoding the enzyme;

(c) a technique for introducing mutation into the enzyme;

(d) a technique for inhibiting transcription and/or translation of agene encoding the enzyme.

(35) The cell according to any one of (23) to (34), which is resistantto at least a lectin which recognizes a sugar chain in which 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in the N-glycoside-linked sugar chain.(36) The cell according to any one of (23) to (35), which is a cellselected from the group consisting of the following (a) to (i):

(a) a CHO cell derived from a Chinese hamster ovary tissue;

(b) a rat myeloma cell line, YB2/3HL.P2.G11.16Ag.20 cell;

(c) a mouse myeloma cell line, NSO cell;

(d) a mouse myeloma cell line, SP2/0-Ag14 cell;

(e) a BHK cell derived from a Syrian hamster kidney tissue;

(f) an antibody-producing hybridoma cell;

(g) a human leukemia cell line, Namalwa cell;

(h) an embryonic stem cell;

(i) a fertilized egg cell.

(37) The cell according to any one of (23) to (36) into which a geneencoding an antibody molecule is introduced.(38) The cell according to (37), wherein the antibody molecule belongsto an IgG class.(39) A method for producing an antibody composition, which comprisesculturing the cell according to (37) or(38) in a medium to produce and accumulate the antibody composition inthe culture; and recovering the antibody composition from the culture.(40) The method according to (39), which produces an antibodycomposition having higher antibody-dependent cell-mediated cytotoxicactivity than an antibody composition obtained from its parent cellline.(41) An antibody composition which is produced using the methodaccording to (39) or (40).(42) A transgenic non-human animal or plant or the progenies thereof,comprising a genome which is modified such that the activity of anenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose and/or the activity of an enzyme relating to the modificationof a sugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in theN-glycoside-linked sugar chain is decreased.(43) The transgenic non-human animal or plant or the progenies thereofaccording to (42), wherein a gene encoding the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or a geneencoding the enzyme relating to the modification of a sugar chain inwhich 1-position of fucose is bound to 6-position of N-acetylglucosaminein the reducing end through lα-bond in the N-glycoside-linked sugarchain is knocked out.(44) The transgenic non-human animal or plant or the progenies thereofaccording to (42) or (43), wherein the enzyme relating to the synthesisof an intracellular sugar nucleotide, GDP-fucose is an enzyme selectedfrom the group consisting of the following (a), (b) and (c):

(a) GMD (GDP-mannose 4,6-dehydratase);

(b) Fx (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase);

(c) GFPP (GDP-beta-L-fucose pyrophosphorylase).

(45) The transgenic non-human animal or plant or the progenies thereofaccording to (44), wherein the GMD is a protein encoded by a DNA of thefollowing (a) or (b):

(a) a DNA comprising the nucleotide sequence represented by SEQ IDNO:65;

(b) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:65 under stringent conditions andencodes a protein having GMD activity.

(46) The transgenic non-human animal or plant or the progenies thereofaccording to (44), wherein the Fx is a protein encoded by a DNA of thefollowing (a) or (b):

(a) a DNA comprising the nucleotide sequence represented by SEQ IDNO:48;

(b) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:48 under stringent conditions andencodes a protein having Fx activity.

(47) The transgenic non-human animal or plant or the progenies thereofaccording to (44), wherein the GFPP is a protein encoded by a DNA of thefollowing (a) or (b):

(a) a DNA comprising the nucleotide sequence represented by SEQ IDNO:51;

(b) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:51 under stringent conditions andencodes a protein having GFPP activity.

(48) The transgenic non-human animal or plant or the progenies thereofaccording to (42) or (43), wherein the enzyme relating to themodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe N-glycoside-linked sugar chain is α-1,6-fucosyltransferase.(49) The transgenic non-human animal or plant or the progenies thereofaccording to (48), wherein the α-1,6-fucosyltransferase is a proteinencoded by a DNA selected from the group consisting of the following(a), (b), (c) and (d):

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1;

(b) a DNA comprising the nucleotide sequence represented by SEQ ID NO:2;

(c) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:1 under stringent conditions andencodes a protein having α-1,6-fucosyltransferase activity;

(d) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:2 under stringent conditions andencodes a protein having α-1,6-fucosyltransferase activity.

(50) The transgenic non-human animal or plant or the progenies thereofaccording to any one of (42) to (49), wherein the transgenic non-humananimal is an animal selected from the group consisting of cattle, sheep,goat, pig, horse, mouse, rat, fowl, monkey and rabbit.(51) A method for producing an antibody composition, which comprisesintroducing a gene encoding an antibody molecule into the transgenicnon-human animal or plant or the progenies thereof according to any oneof (42) to (50); rearing the animal or plant; isolating tissue or bodyfluid comprising the introduced antibody from the reared animal orplant; and recovering the antibody composition from the isolated tissueor body fluid.(52) The method according to (51), wherein the antibody molecule belongsto an IgG class.(53) The method according to (51) or (52), which produces an antibodycomposition having higher antibody-dependent cell-mediated cytotoxicactivity than an antibody composition obtained from a non-human animalor plant or the progenies thereof whose genome is not modified.(54) An antibody composition which is produced using the methodaccording to any one of (51) to (53).(55) A medicament comprising the antibody composition according to anyone of (21), (22), (41) and (54) as an active ingredient.(56) The medicament according to (55), wherein the medicament is adiagnostic drug, a preventive drug or a therapeutic drug for diseasesaccompanied by tumors, diseases accompanied by allergies, diseasesaccompanied by inflammations, autoimmune diseases, circulatory organdiseases, diseases accompanied by viral infections or diseasesaccompanied by bacterial infections.(57) A protein selected from the group consisting of the following (a),(b), (c), (d), (e), (f), (g), (h), (i) and (j)

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:71;

(b) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:71 and has GMD activity;

(c) a protein comprising the amino acid sequence represented by SEQ IDNO:72;

(d) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:72 and has Fx activity;

(e) a protein comprising the amino acid sequence represented by SEQ IDNO:73;

(f) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:73 and has GFPP activity;

(g) a protein comprising the amino acid sequence represented by SEQ IDNO:23;

(h) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:23 and hasα-1,6-fucosyltransferase activity;

(i) a protein comprising the amino acid sequence represented by SEQ IDNO:24;

(j) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:24 and theα-1,6-fucosyltransferase activity.

(58) A DNA which encodes the protein according to (57).(59) A DNA selected from the group consisting of the following (a), (b),(c), (d) and (e):

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1;

(b) a DNA comprising the nucleotide sequence represented by SEQ ID NO:2;

(c) a DNA comprising the nucleotide sequence represented by SEQ IDNO:65;

(d) a DNA comprising the nucleotide sequence represented by SEQ IDNO:48;

(e) a DNA comprising the nucleotide sequence represented by SEQ IDNO:51.

(60) A genome DNA selected from the group consisting of the following(a), (b) and (c):

(a) a genome DNA comprising the nucleotide sequence represented by SEQID NO:3;

(b) a genome DNA comprising the nucleotide sequence represented by SEQID NO:67;

(c) a genome DNA comprising the nucleotide sequence represented by SEQID NO:70.

(61) A target vector for homologous recombination, comprising a fulllength of the DNA according to any one of (58) to (60), or a partthereof.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows electrophoresis patterns of SDS-PAGE of five purifiedanti-GD3 chimeric antibodies (using gradient gel from 4 to 15%). FIG. 1Aand FIG. 1B show a result of the electrophoresis under non-reducingconditions and that under reducing conditions, respectively. Lanes 1 to7 show electrophoresis patterns of high molecular weight markers,YB2/0-GD3 chimeric antibody, CHO/DG44-GD3 chimeric antibody, SP2/0-GD3chimeric antibody, NS0-GD3 chimeric antibody (302), NS0-GD3 chimericantibody (GIT) and low molecular weight markers, respectively.

FIG. 2 shows activities of five purified anti-GD3 chimeric antibodies tobind to GD3, measured by changing the antibody concentration. Theordinate and the abscissa show the binding activity with GD3 and theantibody concentration, respectively. “◯”, “”, “□”, “▪” and “Δ” showthe activities of YB2/0-GD3 chimeric antibody, CHO/DG44-GD3 chimericantibody, SP2/0-GD3 chimeric antibody, NS0-GD3 chimeric antibody (302)and NS0-GD3 chimeric antibody (GIT), respectively.

FIG. 3 shows ADCC activities of five purified anti-GD3 chimericantibodies for a human melanoma cell line G-361. The ordinate andabscissa show the cytotoxic activity and the antibody concentration,respectively. “◯”, “”, “□”, “▪” and “Δ” show the activities ofYB2/0-GD3 chimeric antibody, CHO/DG44-GD3 chimeric antibody, SP2/0-GD3chimeric antibody, NS0-GD3 chimeric antibody (302) and NS0-GD3 chimericantibody (GIT), respectively.

FIG. 4 shows electrophoresis patterns of SDS-PAGE of three purifiedanti-hIL-5Rα CDR-grafted antibodies (using gradient gel from 4 to 15%).FIG. 4A and FIG. 4B show results of the electrophoresis carried outunder non-reducing conditions and those under reducing conditions,respectively. Lanes 1 to 5 show electrophoresis patterns of highmolecular weight markers, YB2/0-hIL-5R CDR antibody, CHO/d-hIL-5R CDRantibody, NS1-hIL-5R CDR antibody and low molecular weight markers,respectively.

FIG. 5 shows activities of three purified anti-hIL-5Rα CDR-graftedantibodies to bind to hIL-5Rα, measured by changing the antibodyconcentration. The ordinate and the abscissa show the binding activitywith hIL-5Rα and the antibody concentration, respectively. “◯”, “” and“□” show the activities of YB2/0-hIL-5R CDR antibody, CHO/d-hIL-5R CDRantibody and NS1-hIL-5R CDR antibody, respectively.

FIG. 6 show ADCC activities of three purified anti-hIL-5Rα CDR-graftedantibodies for an hIL-5R expressing mouse T cell line CTLL-2(h5R). Theordinate and the abscissas show the cytotoxic activity and the antibodyconcentration, respectively. “◯”, “” and “□” show the activities ofYB2/0-hIL-5RCDR antibody, CHO/d-hIL-5R CDR antibody and NS0-hIL-5R CDRantibody, respectively.

FIG. 7 shows inhibition activities of three purified anti-hIL-5RαCDR-grafted antibodies in an hIL-5-induced eosinophil increasing modelof Macaca faseicularis. The ordinate and the abscissa show the number ofeosinophils in peripheral blood and the number of days (the day of thecommencement of antibody and hIL-5 administration was defined as 0 day).“101 and 102”, “301, 302 and 303”, “401, 402 and 403” and “501, 502 and503” show results in the antibody non-administration group, theYB2/0-hIL-5R CDR antibody administered group, the CHO/d-hIL-5R CDRantibody administered group and the NS0-hIL-5R CDR antibody administeredgroup, respectively.

FIG. 8 shows elution patterns of reverse phase HPLC elution of aPA-treated sugar chain (left side), and an elution pattern obtained bytreating the PA-treated sugar chain with α-L-fucosidase and thenanalyzed by reverse phase HPLC (right side), of the purifiedanti-hIL-5Rα CDR-grafted antibody produced by YB2/0 (FIG. 8A) and thepurified anti-hIL-5Rα CDR-grafted antibody produced by NS0 (FIG. 8B).The ordinates and the abscissas show the relative fluorescence intensityand the elution time, respectively.

FIG. 9 shows an elution pattern obtained by preparing a PA-treated sugarchain from the purified anti-hIL-5Rα CDR-grafted antibody produced byCHO/d cell and analyzing it by reverse phase HPLC. The ordinate and theabscissa show the relative fluorescence intensity and the elution time,respectively.

In FIG. 10, FIG. 10A shows the GD3-binding activities of a non-adsorbedfraction and a part of an adsorbed fraction, measured by changing theantibody concentration. The ordinate and the abscissa show the bindingactivity with GD3 and the antibody concentration, respectively. “” and“◯” show the non-adsorbed fraction and a part of the adsorbed fraction,respectively. FIG. 10B shows the ADCC activities of the non-adsorbedfraction and a part of the adsorbed fraction for a human melanoma lineG-361. The ordinate and the abscissa show the cytotoxic activity and theantibody concentration, respectively. “” and “◯” show the non-adsorbedfraction and a part of the adsorbed fraction, respectively.

FIG. 11 shows elution patterns obtained by analyzing PA-treated sugarchains prepared from a non-adsorbed fraction and a part of an adsorbedfraction by a reverse HPLC. FIG. 11A and FIG. 11B show an elutionpattern of the non-adsorbed fraction and an elution pattern of a part ofthe adsorbed fraction, respectively. The ordinates and the abscissasshow the relative fluorescence strength and the elution time,respectively.

FIG. 12 shows elution patterns of PA-treated sugar chains prepared from6 anti-GD3 chimeric antibodies (FIG. 12A to FIG. 12F), obtained byanalyzing them by reverse phase HPLC. The ordinates and the abscissasshow the relative fluorescence intensity and the elution time,respectively.

FIG. 13 shows GD3-binding activities of 6 anti-GD3 chimeric antibodieshaving a different ratio of α-1,6-fucose-free sugar chains measured bychanging the antibody concentration. The ordinate and the abscissa showthe binding activity with GD3 and the antibody concentration,respectively. “”, “□”, “▪”, “Δ”, “▴” and “x” show the activities ofanti-GD3 chimeric antibody (50%), anti-GD3 chimeric antibody (45%),anti-GD3 chimeric antibody (29%), anti-GD3 chimeric antibody (24%),anti-GD3 chimeric antibody (13%) and anti-GD3 chimeric antibody (7%),respectively.

FIG. 14 shows ADCC activities of six kinds of anti-GD3 chimericantibodies having a different ratio of α-1,6-fucose-free sugar chainsagainst a human melanoma cell line G-361, using an effector cell of thedonor A. The ordinate and the abscissa show the cytotoxic activity andthe antibody concentration, respectively. “”, “□”, “▪”, “Δ”, “▴” and“x” show the activities of anti-GD3 chimeric antibody (50%), anti-GD3chimeric antibody (45%), anti-GD3 chimeric antibody (29%), anti-GD3chimeric antibody (24%), anti-GD3 chimeric antibody (13%) and anti-GD3chimeric antibody (7%), respectively.

FIG. 15 shows ADCC activities of six kinds of anti-GD3 chimericantibodies having a different ratio of α-1,6-fucose-free sugar chainsagainst a human melanoma cell line G-361, using an effector cell of thedonor B. The ordinate and the abscissa show the cytotoxic activity andthe antibody concentration, respectively. “”, “□”, “▪”, “Δ”, “▴” and“x” show the activities of anti-GD3 chimeric antibody (50%), anti-GD3chimeric antibody (45%), anti-GD3 chimeric antibody (29%), anti-GD3chimeric antibody (24%), anti-GD3 chimeric antibody (13%) and anti-GD3chimeric antibody (7%), respectively.

FIG. 16 shows elution patterns of PA-treated sugar chains prepared fromsix kinds of anti-CCR4 chimeric antibodies (FIG. 16A to FIG. 16F),obtained by analyzing them by reverse phase HPLC. The ordinates and theabscissas show the relative fluorescence intensity and the elution time,respectively.

FIG. 17 shows CCR4-binding activities of six kinds of anti-CCR4 chimericantibodies having a different ratio of α-1,6-fucose-free sugar chainsmeasured by changing the antibody concentration. The ordinate and theabscissa show the binding activity with CCR4 and the antibodyconcentration, respectively. “▪”, “□”, “▴”, “Δ”, “” and “◯” show theactivities of anti-CCR4 chimeric antibody (46%), anti-CCR4 chimericantibody (39%), anti-CCR4 chimeric antibody (27%), anti-CCR4 chimericantibody (18%), anti-CCR4 chimeric antibody (9%) and anti-CCR4 chimericantibody (8%), respectively.

FIG. 18 shows ADCC activities of anti-CCR4 chimeric antibodies having adifferent ratio of α-1,6-fucose-free sugar chains against CCR4/EL-4cell, using an effector cell of the donor A. The ordinate and theabscissa show the cytotoxic activity and the antibody concentration,respectively. “▪”, “□”, “▴”, “Δ”, “” and “◯” show the activities ofanti-CCR4 chimeric antibody (46%), anti-CCR4 chimeric antibody (39%),anti-CCR4 chimeric antibody (27%), anti-CCR4 chimeric antibody (18%),anti-CCR4 chimeric antibody (9%) and anti-CCR4 chimeric antibody (8%),respectively.

FIG. 19 shows ADCC activities of anti-CCR4 chimeric antibodies having adifferent ratio of α-1,6-fucose-free sugar chain against CCR4/EL-4 cell,using an effector cell of the donor B. The ordinate and the abscissashow the cytotoxic activity and the antibody concentration,respectively. “▪”, “□”, “▴”, “Δ”, “” and “◯” show the activities ofanti-CCR4 chimeric antibody (46%), anti-CCR4 chimeric antibody (39%),anti-CCR4 chimeric antibody (27%), anti-CCR4 chimeric antibody (18%),anti-CCR4 chimeric antibody (9%) and anti-CCR4 chimeric antibody (8%),respectively.

FIG. 20 shows construction of plasmids CHFT8-pCR2.1 and YBFT8-pCR2.1.

FIG. 21 shows construction of plasmids CHAc-pBS and YBAc-pBS.

FIG. 22 shows construction of plasmids CHFT8d-pCR2.1 and YBFT8d-pCR2.1.

FIG. 23 shows construction of plasmids CHAcd-pBS and YBAcd-pBS.

FIG. 24 shows results of determination of an FUT8 transcription productin each host cell line using competitive RT-PCR. Amounts of the FUT8transcription product in each host cell line when rat FUT8 sequence wasused as the standard and internal control are shown. “▪” and “□” showresults when CHO cell line and YB2/0 cell line, respectively, were usedas the host cell.

FIG. 25 shows construction of a plasmid mfFUT8-pCR2.1.

FIG. 26 shows construction of a plasmid pBSmfFUT8.

FIG. 27 shows construction of a plasmid pAGEmfFUT8.

FIG. 28 shows results of analysis of expression levels of FUT8 gene by acell line excessively expressing the gene using a competitive RT-PCR.The ordinate shows relative values of amounts of FUT8 transcription toamounts of β-actin transcription.

FIG. 29 shows ADCC activities of an anti-GD3 chimeric antibody purifiedfrom a cell line excessively expressing FUT8 gene against a humanmelanoma cell line G-361. The ordinate and the abscissa show thecytotoxic activity and the antibody concentration, respectively.

FIG. 30 shows elution patterns of PA-treated sugar chains prepared fromantibodies produced by mfFUT8-6 and pAGE249-introduced cell lines,obtained by analyzing them by reverse phase HPLC. FIG. 30A and FIG. 30Bshow elution patterns of PA-treated sugar chains prepared from anantibody produced by mfFUT8-6-introduced cell line and PA-treated sugarchains prepared from an antibody produced by pAGE249-introduced cellline, respectively. The ordinate and the abscissa show the relativefluorescence intensity and the elution time, respectively.

FIG. 31 shows an elution pattern of PA-treated sugar chains preparedfrom Herceptin, obtained by analyzing them by reverse phase HPLC. Theordinate and the abscissa show the relative fluorescence intensity andthe elution time, respectively.

FIG. 32 shows construction of a plasmid CHfFUT8-pCR2.1.

FIG. 33 shows construction of a plasmid ploxPPuro.

FIG. 34 shows construction of a plasmid pKOFUT8gE2-1.

FIG. 35 shows construction of a plasmid pKOFUT8gE2-2.

FIG. 36 shows construction of a plasmid pscFUT8gE2-3.

FIG. 37 shows construction of a plasmid pKOFUT8gE2-3.

FIG. 38 shows construction of a plasmid pKOFUT8gE2-4.

FIG. 39 shows construction of a plasmid pKOFUT8gE2-5.

FIG. 40 shows construction of a plasmid pKOFUT8Puro.

FIG. 41 shows results of genome Southern analyses of 1st.ΔFUT8 2-46-1and 1st.ΔFUT8 2-46 as α-1,6-fucosyltransferase gene-disrupted CHO celllines.

FIG. 42 shows ADCC activities of an anti-CCR4 chimeric antibody purifiedfrom an FUT8 allele gene-disrupted cell line. The ordinate and theabscissa show the cytotoxic activity and the antibody concentration. “▴”and “▪” show the activities of a purified antibody derived from ananti-CCR4 chimeric antibody-producing CHO cell 5-03 and a purifiedantibody derived from 1st.ΔFUT8 2-46-1, respectively.

FIG. 43 shows ADCC activities of anti-CCR4 human chimeric antibodiesproduced by lectin-resistant cell lines. The ordinate and the abscissashow the cytotoxic activity and the antibody concentration. “□”, “▪”,“♦” and “▴” show the activities of antibodies produced by the strain5-03, CHO/CCR4-LCA, CHO/CCR4-AAL and CHO/CCR4-PHA, respectively.

FIG. 44 shows ADCC activities of anti-CCR4 human chimeric antibodiesproduced by lectin-resistant cell lines. The ordinate and the abscissashow the cytotoxic activity and the antibody concentration,respectively. “□”, “Δ” and “” show activities of antibodies produced byYB2/0 (KM2760 #58-35-16), 5-03 and CHO/CCR4-LCA, respectively.

FIG. 45 shows elution patterns of PA-treated sugar chains prepared frompurified anti-CCR4 human chimeric antibodies, obtained by analyzing themby reverse phase HPLC. The ordinate and the abscissa show the relativefluorescence intensity and the elution time, respectively. FIG. 45A,FIG. 45B, FIG. 45C and FIG. 45D show results of analyses of an antibodyproduced by the strain 5-03, an antibody produced by CHO/CCR4-LCA, anantibody produced by CHO/CCR4-AAL and an antibody produced byCHO/CCR4-PHA, respectively.

FIG. 46 shows the 1st step of construction of an expression vector ofCHO cell-derived GMD (6 steps in total).

FIG. 47 shows the 2nd step of construction of the expression vector ofCHO cell-derived GMD (6 steps in total).

FIG. 48 shows the 3rd step of construction of the expression vector ofCHO cell-derived GMD (6 steps in total).

FIG. 49 shows the 4th step of construction of the expression vector ofCHO cell-derived GMD (6 steps in total).

FIG. 50 shows the 5th step of construction of the expression vector ofCHO cell-derived GMD (6 steps in total).

FIG. 51 shows the 6th step of construction of the expression vector ofCHO cell-derived GMD (6 steps in total).

FIG. 52 shows resistance of GMD-expressed CHO/CCR4-LCA for LCA lectin.The measurement was carried out twice by defining the survival rate of agroup of cells cultured without adding LCA lectin as 100%. In thedrawing, “249” shows the survival rate of the CHO/CCR4-LCA introducedwith an expression vector pAGE249 for LCA lectin. GMD shows resistanceof the CHO/CCR4-LCA introduced with a GMD expression vector pAGE249GMDfor LCA lectin.

FIG. 53 shows ADCC activities of an anti-CCR4 chimeric antibody producedby cells of GMD-expressed CHO/CCR4-LCA cell lines. The ordinate and theabscissa show the cytotoxic activity and the antibody concentration,respectively.

FIG. 54 show a production step of a plasmid CHO-GMD in which the5′-terminal of a clone 34-2 is introduced into the 5′-terminal of a CHOcell-derived GMD cDNA clone 22-8.

FIG. 55 shows an elution pattern of PA-treated sugar chains preparedfrom an anti-CCR4 human chimeric antibody purified from GMDgene-expressed CHO/CCR4-LCA, obtained by analyzing them by reverse phaseHPLC. The ordinate and the abscissa show the relative fluorescenceintensity and the elution time, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The Chinese hamster ovary tissue-derived CHO cell into which a geneencoding an antibody molecule is introduced according to the presentinvention may be any CHO cell, so long as it is a Chinese hamster ovarytissue-derived CHO cell into which a gene encoding an antibody moleculeis introduced, which produces an antibody composition comprising complexN-glycoside-linked sugar chains bound to the Fc region of an antibodymolecule, wherein among the total complex N-glycoside-linked sugarchains bound to the Fc region in the composition, the ratio of a sugarchain in which fucose is not bound to N-acetylglucosamine in thereducing end in the sugar chain is 20% or more.

In the present invention, the antibody molecule includes any molecule,so long as it comprises the Fc region of an antibody. Examples includean antibody, an antibody fragment, a fusion protein comprising an Fcregion, and the like.

The antibody is a protein which is produced in the living body by immunereaction as a result of exogenous antigen stimulation and has anactivity to specifically bind to the antigen. Examples of the antibodyinclude an antibody secreted by a hybridoma cell prepared from a spleencell of an animal immunized with an antigen; an antibody prepared by agenetic recombination technique, namely an antibody obtained byintroducing an antibody gene-inserted antibody expression vector into ahost cell; and the like. Specific examples include an antibody producedby a hybridoma, a humanized antibody, a human antibody and the like.

A hybridoma is a cell which is obtained by cell fusion between a B cellobtained by immunizing a mammal other than human with an antigen and amyeloma cell derived from mouse or the like and can produce a monoclonalantibody having the desired antigen specificity.

Examples of the humanized antibody include a human chimeric antibody, ahuman CDR-grafted antibody and the like.

A human chimeric antibody is an antibody which comprises an antibodyheavy chain variable region (hereinafter referred to as “HV” or “VH”,the heavy chain being “H chain”) and an antibody light chain variableregion (hereinafter referred to as “LV” or “VL”, the light chain being“L chain”), both of an animal other than human, a human antibody heavychain constant region (hereinafter also referred to as “CH”) and a humanantibody light chain constant region (hereinafter also referred to as“CL”). As the animal other than human, any animal such as mouse, rat,hamster, rabbit or the like can be used, so long as a hybridoma can beprepared therefrom.

The human chimeric antibody can be produced by obtaining cDNA's encodingVH and VL from a monoclonal antibody-producing hybridoma, inserting theminto an expression vector for host cell having genes encoding humanantibody CH and human antibody CL to thereby construct a human chimericantibody expression vector, and then introducing the vector into a hostcell to express the antibody.

As the CH of human chimeric antibody, any CH can be used, so long as itbelongs to human immunoglobulin (hereinafter referred to as “hIg”) canbe used. But those belonging to the hIgG class are preferable and anyone of the subclasses belonging to the hIgG class, such as hIgG1, hIgG2,hIgG3 and hIgG4, can be used. Also, as the CL of human chimericantibody, any CL can be used, so long as it belongs to the hIg class,and those belonging to the κ class or λ class can also be used.

A human CDR-grafted antibody is an antibody in which amino acidsequences of CDR's of VH and VL of an antibody derived from an animalother than human are grafted into appropriate positions of VH and VL ofa human antibody.

The human CDR-grafted antibody can be produced by constructing cDNA'sencoding V regions in which CDR's of VH and VL of an antibody derivedfrom an animal other than human are grafted into CDR's of VH and VL of ahuman antibody, inserting them into an expression vector for host cellhaving genes encoding human antibody CH and human antibody CL to therebyconstruct a human CDR-grafted antibody expression vector, and thenintroducing the expression vector into a host cell to express the humanCDR-grafted antibody.

As the CH of human CDR-grafted antibody, any CH can be used, so long asit belongs to the hIg, but those of the hIgG class are preferable andany one of the subclasses belonging to the hIgG class, such as hIgG1,hIgG2, hIgG3 and hIgG4, can be used. Also, as the CL of humanCDR-grafted antibody, any CL can be used, so long as it belongs to thehIg class, and those belonging to the κ class or λ class can also beused.

A human antibody is originally an antibody naturally existing in thehuman body, but it also includes antibodies obtained from a humanantibody phage library, a human antibody-producing transgenic animal anda human antibody-producing transgenic plant, which are prepared based onthe recent advance in genetic engineering, cell engineering anddevelopmental engineering techniques.

Regarding the antibody existing in the human body, a lymphocyte capableof producing the antibody can be cultured by isolating a humanperipheral blood lymphocyte, immortalizing it by its infection with EBvirus or the like and then cloning it, and the antibody can be purifiedfrom the culture.

The human antibody phage library is a library in which antibodyfragments such as Fab, single chain antibody and the like are expressedon the phage surface by inserting a gene encoding an antibody preparedfrom a human B cell into a phage gene. A phage expressing an antibodyfragment having the desired antigen binding activity can be recoveredfrom the library, using its activity to bind to an antigen-immobilizedsubstrate as the marker. The antibody fragment can be converted furtherinto a human antibody molecule comprising two full H chains and two fullL chains by genetic engineering techniques.

A human antibody-producing transgenic non-human animal is an animal inwhich a human antibody gene is introduced into cells. Specifically, ahuman antibody-producing transgenic animal can be prepared byintroducing a human antibody gene into ES cell of a mouse, transplantingthe ES cell into an early stage embryo of other mouse and thendeveloping it. By introducing a human chimeric antibody gene into afertilized egg and developing it, the transgenic animal can be alsoprepared. Regarding the preparation method of a human antibody from thehuman antibody-producing transgenic animal, the human antibody can beproduced and accumulated in a culture by obtaining a humanantibody-producing hybridoma by a hybridoma preparation method usuallycarried out in mammals other than human and then culturing it.

Examples of the transgenic non-human animal include cattle, sheep, goat,pig, horse, mouse, rat, fowl, monkey, rabbit and the like.

Also, in the present invention, it is preferable that the antibody is anantibody which recognizes a tumor-related antigen, an antibody whichrecognizes an allergy- or inflammation-related antigen, an antibodywhich recognizes circulatory organ disease-related antigen, an antibodywhich recognizes an autoimmune disease-related antigen or an antibodywhich recognizes a viral or bacterial infection-related antigen, and ahuman antibody which belongs to the IgG class is preferable.

An antibody fragment is a fragment which comprises the Fc region of anantibody. Examples of the antibody fragment include an H chain monomer,an H chain dimer and the like.

A fusion protein comprising an Fc region is a composition in which anantibody comprising the Fc region of an antibody or the antibodyfragment is fused with a protein such as an enzyme, a cytokine or thelike.

In the present invention, examples of the sugar chain which binds to theFc region of an antibody molecule includes an N-glycoside-linked sugarchain. Examples of the N-glycoside-linked sugar chain include a complextype in which the non-reducing end side of the core structure has one orplural parallel branches of galactose-N-acetylglucosamine (hereinafterreferred to as “Gal-GlcNAc”) and the non-reducing end side of Gal-GlcNAchas a structure such as sialic acid, bisecting N-acetylglucosamine orthe like.

In one antibody, the Fc region has positions to which anN-glycoside-linked sugar chain is bound which will be described later.Accordingly, two sugar chains are bound per one antibody molecule. Sincethe N-glycoside-linked sugar chain which binds to an antibody includesany sugar chain having the core structure represented by the structuralformula (I), a number of combinations of sugar chains may possible forthe two N-glycoside-linked sugar chains which bind to the antibody.Accordingly, identity of substances can be judged from the viewpoint ofthe sugar structure bound to the Fc region.

In the present invention, the composition which comprises an antibodymolecule having complex N-glycoside-linked sugar chains in the Fc region(hereinafter referred to as “the antibody composition of the presentinvention”) may comprise an antibody having the same sugar chainstructure or an antibody having different sugar chain structures, solong as the effect of the present invention is obtained from thecomposition.

In the present invention, the ratio of a sugar chain in which fucose isnot bound to N-acetylglucosamine in the reducing end in the sugar chainamong the total complex N-glycoside-linked sugar chains bound to the Fcregion contained in the antibody composition is a ratio of the number ofa sugar chain in which fucose is not bound to N-acetylglucosamine in thereducing end in the sugar chain to the total number of the complexN-glycoside-linked sugar chains bound to the Fc region contained in thecomposition.

In the present invention, the sugar chain in which fucose is not boundto N-acetylglucosamine in the reducing end in the complexN-glycoside-linked sugar chain is a sugar chain in which the fucose isnot bound to N-acetylglucosamine in the reducing end through α-bond inthe complex N-glycoside-linked sugar chain. Examples include a complexN-glycoside-linked sugar chain in which 1-position of fucose is notbound to 6-position of N-acetylglucosamine through α-bond.

The antibody composition shows high ADCC activity when the ratio of asugar chain in which fucose is not bound to N-acetylglucosamine in thereducing end in the sugar chain among the total complexN-glycoside-linked sugar chains binding to the Fc region contained inthe antibody composition of the present invention is preferably 20% ormore, more preferably 25% or more, still more preferably 30% or more,far preferably 40% or more, and most preferably 50% or more. As theantibody concentration is decreased, the ADCC activity is decreased, buthigh ADCC activity can be obtained even when the antibody concentrationis low, so long as the ratio of a sugar chain in which fucose is notbound to N-acetylglucosamine in the reducing end in the sugar chain is20% or more.

The ratio of a sugar chain in which fucose is not bound toN-acetylglucosamine in the reducing end in the sugar chain contained inthe composition which comprises an antibody molecule having complexN-glycoside-linked sugar chains in the Fc region can be determined byreleasing the sugar chain from the antibody molecule using a knownmethod such as hydrazinolysis, enzyme digestion or the like [BiochemicalExperimentation Methods 23—Method for Studying Glycoprotein Sugar Chain(Japan Scientific Societies Press), edited by Reiko Takahashi (1989)],carrying out fluorescence labeling or radioisotope labeling of thereleased sugar chain and then separating the labeled sugar chain bychromatography. Also, the released sugar chain can also be determined byanalyzing it with the HPAED-PAD method [J. Liq. Chromatogr., 6, 1557(1983)].

In the present invention, the Chinese hamster ovary tissue-derived CHOcell includes any cell which is a cell line established from an ovarytissue of Chinese hamster (Cricetulus griseus). Examples include CHOcells described in documents such as Journal of Experimental Medicine,108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics,55, 513 (1968); Chromosoma, 41, 129 (1973); Methods in Cell Science, 18,115 (1996); Radiation Research, 148, 260 (1997); Proc. Natl. Acad. Sci.USA, 77, 4216 (1980); Proc. Natl. Acad. Sci., 60, 1275 (1968); Cell, 6,121 (1975); Molecular Cell Genetics, Appendix I, II (pp. 883-900); andthe like. In addition, CHO-K1 (ATCC CCL-61), DUXB11 (ATCC CCL-9096) andPro-5 (ATCC CCL-1781) registered in ATCC (The American Type CultureCollection) and a commercially available CHO-S (Life Technologies, Cat#11619) or sub-cell lines obtained by adapting the cell lines usingvarious media can also be exemplified.

In the present invention, the enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose may be any enzyme, so long asit is an enzyme relating to the synthesis of the intracellular sugarnucleotide, GDP-fucose as a supply source of fucose to a sugar chain.The enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose is an enzyme which has influence on the synthesisof the intracellular sugar nucleotide, GDP-fucose.

The intracellular sugar nucleotide, GDP-fucose is supplied by a de novosynthesis pathway or a salvage synthesis pathway. Thus, all enzymesrelating to the synthesis pathways are included in the enzyme relatingto the synthesis of an intracellular sugar nucleotide, GDP-fucose.

Examples of the enzyme relating to the de novo synthesis pathway of theintracellular sugar nucleotide, GDP-fucose include GDP-mannose4,6-dehydratase (hereinafter referred to as “GMD”),GDP-keto-6-deoxymannose 3,5-epimerase 4,6-reductase (hereinafterreferred to as “Fx”) and the like.

Examples of the enzyme relating to the salvage synthesis pathway of theintracellular sugar nucleotide, GDP-fucose include GDP-beta-L-fucosepyrophosphorylase (hereinafter referred to as “GFPP”), fucokinase andthe like.

As the enzyme which has influence on the synthesis of an intracellularsugar nucleotide, GDP-fucose, an enzyme which has influence on theactivity of the enzyme relating to the synthesis of the intracellularsugar nucleotide, GDP-fucose and an enzyme which has influence on thestructure of substances as the substrate of the enzyme are alsoincluded.

In the present invention, examples of the GMD include:

a protein encoded by a DNA of the following (a) or (b):

(a) a DNA comprising the nucleotide sequence represented by SEQ IDNO:65;

(b) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:65 under stringent conditions andencodes a protein having GMD activity,

(c) a protein comprising the amino acid sequence represented by SEQ IDNO:71,

(d) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:71 and has GMD activity,

(e) a protein which comprises an amino acid sequence having a homologyof at least 80% with the amino acid sequence represented by SEQ ID NO:71and has GMD activity, and the like.

Also, examples of the DNA encoding the amino acid sequence of GMDinclude a DNA comprising the nucleotide sequence represented by SEQ IDNO:65 and a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:65 under stringent conditions andencodes an amino acid sequence having GMD activity.

In the present invention, examples of the Fx include:

a protein encoded by a DNA of the following (a) or (b):

(a) a DNA comprising the nucleotide sequence represented by SEQ IDNO:48;

(b) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:48 under stringent conditions andencodes a protein having Fx activity,

(c) a protein comprising the amino acid sequence represented by SEQ IDNO:72,

(d) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:72 and has Fx activity,

(e) a protein which comprises an amino acid sequence having a homologyof at least 80% with the amino acid sequence represented by SEQ ID NO:72and has Fx activity, and the like.

Also, examples of the DNA encoding the amino acid sequence of Fx includea DNA comprising the nucleotide sequence represented by SEQ ID NO:48 anda DNA which hybridizes with the DNA comprising the nucleotide sequencerepresented by SEQ ID NO:48 under stringent conditions and encodes anamino acid sequence having Fx activity.

In the present invention, examples of the GFPP include: a proteinencoded by a DNA of the following (a) or (b):

(a) a DNA comprising the nucleotide sequence represented by SEQ IDNO:51;

(b) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:51 under stringent conditions andencodes a protein having GFPP activity,

(c) a protein comprising the amino acid sequence represented by SEQ IDNO:73,

(d) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:73 and has GFPP activity,

(e) a protein which comprises an amino acid sequence having a homologyof at least 80% with the amino acid sequence represented by SEQ ID NO:73and has GFPP activity, and the like.

Also, examples of the DNA encoding the amino acid sequence of GFPPinclude a DNA comprising the nucleotide sequence represented by SEQ IDNO:51 and a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:51 under stringent conditions andencodes an amino acid sequence having Fx activity.

In the present invention, the enzyme relating to the modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain includes any enzyme, so long as it is anenzyme relating to the reaction of binding of 1-position of fucose to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex N-glycoside-linked sugar chain.

Examples of the enzyme relating to the reaction of binding of 1-positionof fucose to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex N-glycoside-linked sugar chain includeα-1,6-fucosyltransferase, α-L-fucosidase and the like.

Also, examples include an enzyme which has influence on the activity theenzyme relating to the reaction of binding of 1-position of fucose to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex N-glycoside-linked sugar chain and an enzyme which hasinfluence on the structure of substances as the substrate of the enzyme.

In the present invention, examples of the α-1,6-fucosyltransferaseinclude:

a protein encoded by a DNA of the following (a), (b), (c) or (d):

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1;

(b) a DNA comprising the nucleotide sequence represented by SEQ ID NO:2;

(c) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:1 under stringent conditions andencodes a protein having α-1,6-fucosyltransferase activity;

(d) a DNA which hybridizes with the DNA comprising the nucleotidesequence represented by SEQ ID NO:2 under stringent conditions andencodes a protein having α-1,6-fucosyltransferase activity;

(e) a protein comprising the amino acid sequence represented by SEQ IDNO:23,

(f) a protein comprising the amino acid sequence represented by SEQ IDNO:24,

(g) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:23 and hasα-1,6-fucosyltransferase activity,

(h) a protein which comprises an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:24 and hasα-1,6-fucosyltransferase activity,

(i) a protein which comprises an amino acid sequence having a homologyof at least 80% with the amino acid sequence represented by SEQ ID NO:23and has α-1,6-fucosyltransferase activity,

(j) a protein which comprises an amino acid sequence having a homologyof at least 80% with the amino acid sequence represented by SEQ ID NO:24and has α-1,6-fucosyltransferase activity, and the like.

Also, examples of the DNA encoding the amino acid sequence ofα-1,6-fucosyltransferase include a DNA having the nucleotide sequencerepresented by SEQ ID NO:1 or 2 and a DNA which hybridizes with the DNAhaving the nucleotide sequence represented by SEQ ID NO:1 or 2 understringent conditions and encodes an amino acid sequence havingα-1,6-fucosyltransferase activity.

In the present invention, a DNA which hybridizes under stringentconditions is a DNA obtained, e.g., by a method such as colonyhybridization, plaque hybridization or Southern blot hybridization usinga DNA such as the DNA having the nucleotide sequence represented by SEQID NO:1, 2, 48, 51 or 65 or a partial fragment thereof as the probe, andspecifically includes a DNA which can be identified by carrying outhybridization at 65° C. in the presence of 0.7 to 1.0 M sodium chlorideusing a filter to which colony- or plaque-derived DNA fragments areimmobilized, and then washing the filter at 65° C. using 0.1 to 2×SSCsolution (composition of the 1×SSC solution comprising 150 mM sodiumchloride and 15 mM sodium citrate). The hybridization can be carried outin accordance with the methods described, e.g., in Molecular Cloning, ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989)(hereinafter referred to as “Molecular Cloning, Second Edition”),Current Protocols in Molecular Biology, John Wiley & Sons, 1987-1997(hereinafter referred to as “Current Protocols in Molecular Biology”);DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition,Oxford University (1995); and the like. Examples of the hybridizable DNAinclude a DNA having at least 60% or more, preferably 70% or more, morepreferably 80% or more, still more preferably 90% or more, far morepreferably 95% or more, and most preferably 98% or more, of homologywith the nucleotide sequence represented by SEQ ID NO:1, 2, 48, 51 or65.

In the present invention, the protein which comprises an amino acidsequence in which at least one amino acid is deleted, substituted,inserted and/or added in the amino acid sequence represented by SEQ IDNO:23, 24, 71, 72 or 73 and has α-1,6-fucosyltransferase activity, GMDactivity, Fx activity or GFPP activity can be obtained, e.g., byintroducing a site-directed mutation into a DNA encoding a proteinhaving the amino acid sequence represented by SEQ ID NO:1, 2, 65, 48 or51, respectively, using the site-directed mutagenesis described, e.g.,in Molecular Cloning, Second Edition; Current Protocols in MolecularBiology; Nucleic Acids Research, 10, 6487 (1982); Proc. Natl. Acad. Sci.USA, 79, 6409 (1982); Gene, 34, 315 (1985); Nucleic Acids Research, 13,4431 (1985); Proc. Natl. Acad. Sci. USA, 82, 488 (1985); and the like.The number of amino acids to be deleted, substituted, inserted and/oradded is one or more, and the number is not particularly limited, but isa number which can be deleted, substituted or added by a known techniquesuch as the site-directed mutagenesis, e.g., it is 1 to several tens,preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5.

Also, in order to maintain the α-1,6-fucosyltransferase activity, GMDactivity, Fx activity or GFPP activity of the protein to be used in thepresent invention, it has at least 80% or more, preferably 85% or more,more preferably 90% or more, still more preferably 95% or more, far morepreferably 97% or more, and most preferably 99% or more, of homologywith the amino acid sequence represented by SEQ ID NO:23, 24, 71, 72 or73, when calculated using an analyzing soft such as BLAST [J. Mol.Biol., 215, 403 (1990)], FASTA [Methods in Enzymology, 183, 63 (1990)]or the like.

Examples of the CHO cell of the present invention include a cell inwhich the enzyme activity is decreased or deleted.

The cell in which the enzyme activity is decreased or deleted includecells in which the activity of an enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or the activity of an enzymerelating to the modification of a sugar chain wherein 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex N-glycoside-linked sugar chain isdecreased or deleted. As the method for obtaining such cells, anytechnique can be used, so long as it can reduce or delete the enzymeactivity of interest. Examples of the technique for reducing or deletingthe enzyme activity include:

(a) a gene disruption technique targeting a gene encoding the enzyme,

(b) a technique for introducing a dominant negative mutant of a geneencoding the enzyme,

(c) a technique for introducing mutation into the enzyme,

(d) a technique for inhibiting transcription and/or translation of agene encoding the enzyme,

(e) a technique for selecting a cell line resistant to a lectin whichrecognizes a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex N-glycoside-linked sugar chain, and the like.

Herein, the lectin-resistant cell line can be obtained by culturing acell line in a medium comprising a predetermined concentration of lectinand then by selecting a cell line which acquires such a property thatits survival rate is increased at least 2 times, preferably 3 times, andmore preferably 5 times or more, than the parent cell line withstatistical significance. Also, it can also be obtained by culturing acell line in a medium comprising lectin and then by selecting a cellline which can be cultured at a certain survival rate, e.g., 80%survival rate, at a lectin concentration of at least 2 times, preferably5 times, more preferably 10 times, and most preferably 20 times or more,than the parent cell line.

As the lectin which recognizes a sugar chain structure in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the N-glycoside-linked sugar chain,any lectin which can recognize the sugar chain structure can be used.Examples include a Lens culinaris lectin LCA (lentil agglutinin derivedfrom Lens culinaris), a pea lectin PSA (pea lectin derived from Pisumsativum), a broad bean lectin VFA (agglutinin derived from Vicia faba),an Aleuria aurantia lectin AAL (lectin derived from Aleuria aurantia)and the like.

The CHO cell of the present invention can produce an antibodycomposition having higher ADCC activity than that of an antibodycomposition produced by the parent CHO cell before applying thetechnique for decreasing or deleting the enzyme activity of interest.

Also, the CHO cell of the present invention can produce an antibodycomposition having higher ADCC activity than that of an antibodycomposition in which, among the total complex N-glycoside-linked sugarchains bound to the Fc region contained in the antibody composition, theratio of a sugar chain in which fucose is not bound toN-acetylglucosamine in the reducing end in the sugar chain is less than20%.

An example of the parent cell line to be used in the present inventionis a cell in which the activity of an enzyme relating to the synthesisof an intracellular sugar nucleotide, GDP-fucose or the activity of anenzyme relating to the modification of a sugar chain wherein 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in the complex N-glycoside-linked sugar chain is notdecreased. Specifically, a cell which is not treated to decrease ordelete the activity of an enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or the activity of an enzymerelating to the modification of a sugar chain wherein 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex N-glycoside-linked sugar chain is used.

In the present invention, the ADCC activity is a cytotoxic activity inwhich an antibody bound to a cell surface antigen on a tumor cell in theliving body activate an effector cell through an Fc receptor existing onthe antibody Fc region and effector cell surface and thereby obstructthe tumor cell and the like [Monoclonal Antibodies: Principles andApplications, Wiley-Liss, Inc., Chapter 2.1 (1955)]. Examples of theeffector cell include a killer cell, a natural killer cell, an activatedmacrophage and the like.

The present invention also relates to a cell in which the activity of anenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the activity of an enzyme relating to the modification ofa sugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain is decreased by a genetic engineeringtechnique (hereinafter referred to as “the host cell of the presentinvention”). The host cell of the present invention is useful as a hostcell for producing an antibody composition having high ADCC activity.

The host cell of the present invention may be any host, so long as itcan express an antibody molecule. Examples include a yeast cell, ananimal cell, an insect cell, a plant cell and the like. Examples of thecells include those which will be later in the item 3. Among animalcells, preferred examples include a CHO cell derived from a Chinesehamster ovary tissue, a rat myeloma cell line YB2/3HL.P2.G11.16Ag.20cell, a mouse myeloma cell line NSO cell, a mouse myeloma SP2/0-Ag14cell, a BHK cell derived from a syrian hamster kidney tissue, anantibody producing-hybridoma cell, a human leukemia cell line Namalwacell, an embryonic stem cell, a fertilized egg cell and the like.

The present invention is described below in detail.

1. Preparation of the Host Cell of the Present Invention

The host cell of the present invention can be prepared by the followingtechniques.

(1) Gene Disruption Technique Targeting at a Gene Encoding an Enzyme

The host cell of the present invention can be prepared using a genedisruption technique by targeting at an enzyme relating to the synthesisof an intracellular sugar nucleotide, GDP-fucose or an enzyme relatingto the modification of a sugar chain wherein 1-position of fucose isbound to 6-position of N-acetylglucosamine in the reducing end throughα-bond in the complex N-glycoside-linked sugar chain. Examples of theenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose include GMD, Fx, GFPP, fucokinase and the like. Examples ofthe enzyme relating to the modification of a sugar chain wherein1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the complex N-glycoside-linked sugarchain include α-1,6-fucosyltransferase, α-L-fucosidase and the like.

The gene as used herein includes DNA and RNA.

The gene disruption method may be any method, so long as it can disruptthe gene of the target enzyme is included. Examples include an antisensemethod, a ribozyme method, a homologous recombination method, an RDOmethod, an RNAi method, a retrovirus-employed method, atransposon-employed method and the like. The methods are specificallydescribed below.

(a) Preparation of the Host Cell of the Present Invention by theAntisense Method or the Ribozyme Method

The host cell of the present invention can be prepared by the ribozymemethod described in Cell Technology, 12, 239 (1993); BIO/TECHNOLOGY, 17,1097 (1999); Hum. Mol. Genet., 5, 1083 (1995); Cell Technology, 13, 255(1994); Proc. Natl. Acad. Sci. USA, 96, 1886 (1999); or the like, e.g.,in the following manner by targeting at an enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or an enzymerelating to the modification of a sugar chain wherein 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex N-glycoside-linked sugar chain.

A cDNA or a genome DNA encoding an enzyme relating to the synthesis ofan intracellular sugar nucleotide, GDP-fucose or an enzyme relating tothe modification of a sugar chain wherein 1-position of fucose is boundto 6-position of N-acetylglucosamine in the reducing end through α-bondin the complex N-glycoside-linked sugar chain is prepared.

The nucleotide sequence of the prepared cDNA or genome DNA isdetermined.

Based on the determined DNA sequence, an appropriate length of anantisense gene or ribozyme construct comprising a DNA moiety whichencodes the enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose or the enzyme relating to the modification of asugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain a part of its non-translation region oran intron, is designed.

In order to express the antisense gene or ribozyme in a cell, arecombinant vector is prepared by inserting a fragment or total lengthof the prepared DNA into downstream of the promoter of an appropriateexpression vector.

A transformant is obtained by introducing the recombinant vector into ahost cell suitable for the expression vector.

The host cell of the present invention can be obtained by selecting atransformant using, as a marker, the activity of the enzyme relating tothe synthesis of an intracellular sugar nucleotide, GDP-fucose or theenzyme relating to the modification of a sugar chain wherein 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in the complex N-glycoside-linked sugar chain. Thehost cell of the present invention can also be obtained by selecting atransformant as a measure of the sugar chain structure of a glycoproteinon the cell membrane or the sugar chain structure of the producedantibody molecule.

As the host cell used for the production of the host cell of the presentinvention, any cell such as yeast, animal cell, insect cell or plantcell can be used, so long as it has a gene encoding the target enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the target enzyme relating to the modification of a sugarchain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain. Examples include host cells which willbe described later in the item 3.

As the expression vector, a vector which is autonomously replicable inthe host cell or can be integrated into the chromosome and comprises apromoter at such a position that the designed antisense gene or ribozymecan be transferred is used. Examples include expression vectors whichwill be described later in the item 3.

Regarding the method for introducing a gene into various host cells, themethods for introducing recombinant vectors suitable for various hostcells, which will be described later in the item 3, can be used.

The following method can be exemplified as the method for selecting atransformant as a measure of the activity of an enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or theactivity of an enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain.

Method for Selecting Transformant:

Examples of the method for selecting a cell in which the activity of anenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the activity of an enzyme relating to the modification ofa sugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain is decreased include biochemical methodsor genetic engineering techniques described in New BiochemicalExperimentation Series 3-Saccharides I, Glycoprotein (Tokyo KagakuDojin), edited by Japanese Biochemical society (1988); Cell Engineering,Supplement, Experimental Protocol Series, Glycobiology ExperimentalProtocol, Glycoprotein, Glycolipid and Proteoglycan (Shujun-sha), editedby Naoyuki Taniguchi, Akemi Suzuki, Kiyoshi Furukawa and KazuyukiSugawara (1996); Molecular Cloning, Second Edition; Current Protocols inMolecular Biology; and the like. Examples of the biochemical methodinclude a method in which the enzyme activity is evaluated using anenzyme-specific substrate and the like. Examples of the geneticengineering technique include the Northern analysis, RT-PCR and the likewhich measures the amount of mRNA of a gene encoding the enzyme.

Examples of the method for selecting a transformant using the sugarchain structure of a glycoprotein on the cell membrane as a markerinclude the methods which will be described later in the item 1(5).Examples of the method for selecting a transformant using the sugarchain structure of a produced antibody molecule as a marker include themethods which will be described later in the items 5 and 6.

As the method for preparing cDNA encoding an enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or an enzymerelating to the modification of a sugar chain wherein 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex N-glycoside-linked sugar chain, thefollowing method is exemplified.

Preparation of DNA:

A total RNA or mRNA is prepared from a human or non-human animal tissueor cell.

A cDNA library is prepared from the prepared total RNA or mRNA.

Degenerative primers are produced based on the amino acid sequence of anenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or an enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain, and a gene fragment encoding the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain is obtained by PCR using the preparedcDNA library as the template.

A DNA encoding the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the enzyme relating to the modificationof a sugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain can be obtained by screening the cDNAlibrary using the obtained gene fragment as a probe.

Regarding the mRNA of a human or non-human tissue or cell, acommercially available product (e.g., manufactured by Clontech) may beused or it may be prepared from a human or non-human animal tissue orcell in the following manner. Examples of the method for preparing atotal RNA from a human or non-human animal tissue or cell include theguanidine thiocyanate-cesium trifluoroacetate method [Methods inEnzymology, 154, 3 (1987)], the acidic guanidine thiocyanate phenolchloroform (AGPC) method [Analytical Biochemistry, 162, 156 (1987);Experimental Medicine, 9, 1937 (1991)] and the like.

Also, examples of the method for preparing mRNA from a total RNA aspoly(A)⁺ RNA include an oligo(dT)-immobilized cellulose column method(Molecular Cloning, Second Edition) and the like.

In addition, mRNA can be prepared using a kit such as Fast Track mRNAIsolation Kit (manufactured by Invitrogen), Quick Prep mRNA PurificationKit (manufactured by Pharmacia) or the like.

A cDNA library is prepared from the prepared mRNA of a human ornon-human animal tissue or cell. Examples of the method for preparingcDNA libraries include the methods described in Molecular Cloning,Second Edition; Current Protocols in Molecular Biology; A LaboratoryManual, Second Edition (1989); and the like, or methods usingcommercially available kits such as SuperScript Plasmid System for cDNASynthesis and Plasmid Cloning (manufactured by Life Technologies),ZAP-cDNA Synthesis Kit (manufactured by STRATAGENE) and the like.

As the cloning vector for use in the preparation of the cDNA library,any vector such as a phage vector, a plasmid vector or the like can beused, so long as it is autonomously replicable in Escherichia coli K12.Examples include ZAP Express [manufactured by STRATAGENE, Strategies, 5,58 (1992)], pBluescript II SK(+) [Nucleic Acids Research, 17, 9494(1989)], Lambda ZAP II (manufactured by STRATAGENE), λgt10 and λgt11[DNA Cloning, A Practical Approach, 1, 49 (1985)], λTriplEx(manufactured by Clontech), λExCell (manufactured by Pharmacia), pcD2[Mol. Cell. Biol., 3, 280 (1983)], pUC18 [Gene, 33, 103 (1985)] and thelike.

Any microorganism can be used as the host microorganism, but Escherichiacoli is preferably used. Examples include Escherichia coli XL1-Blue MRF′[manufactured by STRATAGENE, Strategies, 5, 81 (1992)], Escherichia coliC600 [Genetics, 39, 440 (1954)], Escherichia coli Y1088 [Science, 222,778 (1983)], Escherichia coli Y1090 [Science, 222, 778 (1983)],Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)], Escherichia coliK802 [J. Mol. Biol., 16, 118 (1966)], Escherichia coli JM105 [Gene, 38,275 (1985)] and the like

The cDNA library may be used as such in the succeeding analysis, and inorder to obtain a full length cDNA as efficient as possible bydecreasing the ratio of an infull length cDNA, a cDNA library preparedusing the oligo cap method developed by Sugano et al. [Gene, 138, 171(1994); Gene, 200, 149 (1997); Protein, Nucleic Acid and Protein, 41,603 (1996); Experimental Medicine, 11, 2491 (1993); cDNA Cloning(Yodo-sha) (1996); Methods for Preparing Gene Libraries (Yodo-sha)(1994)] may be used in the following analysis.

Degenerative primers specific for the 5′-terminal and 3′-terminalnucleotide sequences of a nucleotide sequence presumed to encode theamino acid sequence are prepared based on the amino acid sequence of theenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain, and DNA is amplified by PCR [PCRProtocols, Academic Press (1990)] using the prepared cDNA library as thetemplate to obtain a gene fragment encoding the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or the enzymerelating to the modification of a sugar chain wherein 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex N-glycoside-linked sugar chain.

It can be confirmed that the obtained gene fragment is a DNA encodingthe enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose or the enzyme relating to the modification of asugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain, by a method usually used for analyzing anucleotide, such as the dideoxy method of Sanger et al. [Proc. Natl.Acad. Sci. USA, 74, 5463 (1977)], a nucleotide sequence analyzer such asABIPRISM 377 DNA Sequencer (manufactured by PE Biosystems) or the like.

A DNA encoding the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the enzyme relating to the modificationof a sugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain can be obtained by carrying out colonyhybridization or plaque hybridization (Molecular Cloning, SecondEdition) for the cDNA or cDNA library synthesized from the mRNAcontained in the human or non-human animal tissue or cell, using thegene fragment as a DNA probe.

Also, a DNA encoding the enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or the enzyme relating to themodification of a sugar chain wherein 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex N-glycoside-linked sugar chain can also be obtained bycarrying out screening by PCR using the cDNA or cDNA library synthesizedfrom the mRNA contained in a human or non-human animal tissue or cell asthe template and using the primers used for obtaining the gene fragmentencoding the enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose or the enzyme relating to the modification of asugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain.

The nucleotide sequence of the obtained DNA encoding the enzyme relatingto the synthesis of an intracellular sugar nucleotide, GDP-fucose or theenzyme relating to the modification of a sugar chain wherein 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in the complex N-glycoside-linked sugar chain isanalyzed from its terminus and determined by a method usually used foranalyzing a nucleotide, such as the dideoxy method of Sanger et al.[Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)], a nucleotide sequenceanalyzer such as ABIPRISM 377 DNA Sequencer (manufactured by PEBiosystems) or the like.

A gene encoding the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the enzyme relating to the modificationof a sugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain can also be determined from genes in databases by searching nucleotide sequence data bases such as GenBank, EMBL,DDBJ and the like using a homology retrieving program such as BLASTbased on the determined cDNA nucleotide sequence.

Examples of the nucleotide sequence of the gene obtained by the methodencoding the enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose include the nucleotide sequence represented bySEQ ID NO:48, 51 or 65. Examples of the nucleotide sequence of the geneencoding the enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain include the nucleotide sequencerepresented by SEQ ID NO:1 or 2.

The cDNA encoding the enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or the enzyme relating to themodification of a sugar chain wherein 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex N-glycoside-linked sugar chain can also be obtained bychemically synthesizing it with a DNA synthesizer such as DNASynthesizer model 392 manufactured by Perkin Elmer or the like using thephosphoamidite method, based on the determined DNA nucleotide sequence.

As an example of the method for preparing a genome DNA encoding theenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain, the method described below isexemplified.

Preparation of Genome DNA:

Examples of the method for preparing genome DNA include known methodsdescribed in Molecular Cloning, Second Edition; Current Protocols inMolecular Biology; and the like. In addition, a genome DNA encoding theenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain can also be isolated using a kit such asGenome DNA Library Screening System (manufactured by Genome Systems),Universal GenomeWalker™ Kits (manufactured by CLONTECH) or the like.

Examples of the nucleotide sequence of the genome DNA obtained by themethod encoding the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose include the nucleotide sequence representedby SEQ ID NO:67 or 70. Examples of the nucleotide sequence of the genomeDNA encoding the enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain include the nucleotide sequencerepresented by SEQ ID NO:3.

In addition, the host cell of the present invention can also be obtainedwithout using an expression vector, by directly introducing an antisenseoligonucleotide or ribozyme into a host cell, which is designed based onthe nucleotide sequence encoding the enzyme relating to the synthesis ofan intracellular sugar nucleotide, GDP-fucose or the enzyme relating tothe modification of a sugar chain wherein 1-position of fucose is boundto 6-position of N-acetylglucosamine in the reducing end through α-bondin the complex N-glycoside-linked sugar chain.

The antisense oligonucleotide or ribozyme can be prepared in the usualmethod or using a DNA synthesizer. Specifically, it can be preparedbased on the sequence information of an oligonucleotide having acorresponding sequence of continued 5 to 150 bases, preferably 5 to 60bases, and more preferably 10 to 40 bases, among nucleotide sequences ofa cDNA and a genome DNA encoding the enzyme relating to the synthesis ofan intracellular sugar nucleotide, GDP-fucose or the enzyme relating tothe modification of a sugar chain wherein 1-position of fucose is boundto 6-position of N-acetylglucosamine in the reducing end through α-bondin the complex N-glycoside-linked sugar chain, by synthesizing anoligonucleotide which corresponds to a sequence complementary to theoligonucleotide (antisense oligonucleotide) or a ribozyme comprising theoligonucleotide sequence.

Examples of the oligonucleotide include oligo RNA and derivatives of theoligonucleotide (hereinafter referred to as “oligonucleotidederivatives”).

Examples of the oligonucleotide derivatives include oligonucleotidederivatives in which a phosphodiester bond in the oligonucleotide isconverted into a phosphorothioate bond, an oligonucleotide derivative inwhich a phosphodiester bond in the oligonucleotide is converted into anN3′-P5′ phosphoamidate bond, an oligonucleotide derivative in whichribose and a phosphodiester bond in the oligonucleotide are convertedinto a peptide-nucleic acid bond, an oligonucleotide derivative in whichuracil in the oligonucleotide is substituted with C-5 propynyluracil, anoligonucleotide derivative in which uracil in the oligonucleotide issubstituted with C-5 thiazoleuracil, an oligonucleotide derivative inwhich cytosine in the oligonucleotide is substituted with C-5propynylcytosine, an oligonucleotide derivative in which cytosine in theoligonucleotide is substituted with phenoxazine-modified cytosine, anoligonucleotide derivative in which ribose in the oligonucleotide issubstituted with 2′-O-propylribose and an oligonucleotide derivative inwhich ribose in the oligonucleotide is substituted with2′-methoxyethoxyribose [Cell Technology, 16, 1463 (1997)].

(b) Preparation of the Host Cell of the Present Invention by HomologousRecombination

The host cell of the present invention can be produced by modifying atarget gene on chromosome through a homologous recombination technique,using a gene encoding an enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or an enzyme relating to themodification of a sugar chain wherein 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex N-glycoside-linked sugar chain as the target gene.

The target gene on the chromosome can be modified by using a methoddescribed in Manipulating the Mouse Embryo, A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press (1994) (hereinafterreferred to as “Manipulating the Mouse Embryo, A Laboratory Manual”);Gene Targeting, A Practical Approach, IRL Press at Oxford UniversityPress (1993); Biomanual Series 8, Gene Targeting, Preparation of MutantMice using ES Cells, Yodo-sha (1995) (hereinafter referred to as“Preparation of Mutant Mice using ES Cells”); or the like, for example,as follows.

A genome DNA encoding an enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or an enzyme relating to themodification of a sugar chain wherein 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex N-glycoside-linked sugar chain is prepared.

Based on the nucleotide sequence of the genome DNA, a target vector isprepared for homologous recombination of a target gene to be modified(e.g., structural gene of the enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or the enzyme relating to themodification of a sugar chain wherein 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex N-glycoside-linked sugar chain, or a promoter gene).

The host cell of the present invention can be produced by introducingthe prepared target vector into a host cell and selecting a cell inwhich homologous recombination occurred between the target gene andtarget vector.

As the host cell, any cell such as yeast, animal cell, insect cell orplant cell can be used, so long as it has a gene encoding the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain. Examples include the host cells whichwill be described later in the item 3.

Examples of the method for preparing a genome DNA encoding the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain include the methods described in thepreparation of genome DNA in the item 1(1)(a) and the like.

Examples of the nucleotide sequence of genome DNA encoding the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose include the nucleotide sequence represented by SEQ ID NO:67or 70. Examples of the nucleotide sequence of genome DNA encoding theenzyme relating to the modification of a sugar chain wherein 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in the complex N-glycoside-linked sugar chain includethe nucleotide sequence represented by SEQ ID NO:3.

The target vector for use in the homologous recombination of the targetgene can be prepared in accordance with a method described in GeneTargeting, A Practical Approach, IRL Press at Oxford University Press(1993); Biomanual Series 8, Gene Targeting, Preparation of Mutant Miceusing ES Cells, Yodo-sha (1995); or the like. The target vector can beused as either a replacement type or an insertion type.

For introducing the target vector into various host cells, the methodsfor introducing recombinant vectors suited for various host cells, whichwill be described later in the item 3, can be used.

Examples of the method for efficiently selecting a homologousrecombinant include a method such as the positive selection, promoterselection, negative selection or polyA selection described in GeneTargeting, A Practical Approach, IRL Press at Oxford University Press(1993); Biomanual Series 8, Gene Targeting, Preparation of Mutant Miceusing ES Cells, Yodo-sha (1995); or the like. Examples of the method forselecting the homologous recombinant of interest from the selected celllines include the Southern hybridization method for genome DNA(Molecular Cloning, Second Edition), PCR [PCR Protocols, Academic Press(1990)], and the like.

(c) Preparation of the Host Cell of the Present Invention by RDO Method

The host cell of the present invention can be prepared by an RDO(RNA-DNA oligonucleotide) method by targeting at a gene encoding anenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or an enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain, for example, as follows

A cDNA or a genome DNA encoding an enzyme relating to the synthesis ofan intracellular sugar nucleotide, GDP-fucose or an enzyme relating tothe modification of a sugar chain wherein 1-position of fucose is boundto 6-position of N-acetylglucosamine in the reducing end through α-bondin the complex N-glycoside-linked sugar chain is prepared.

The nucleotide sequence of the prepared cDNA or genome DNA isdetermined.

Based on the determined DNA sequence, an appropriate length of an RDOconstruct comprising a DNA moiety which encodes the enzyme relating tothe synthesis of an intracellular sugar nucleotide, GDP-fucose or theenzyme relating to the modification of a sugar chain wherein 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in the complex N-glycoside-linked sugar chain or apart of its non-translation region or an intron, is designed andsynthesized.

The host cell of the present invention can be obtained by introducingthe synthesized RDO into a host cell and then selecting a transformantin which a mutation occurred in the target enzyme, namely the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain.

As the host cell, any cell such as yeast, animal cell, insect cell orplant cell can be used, so long as it has a gene encoding the targetenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or of the target enzyme relating to the modification of asugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain. Examples include the host cells whichwill be described later in the item 3.

Examples of the method for introducing RDO into various host cellsinclude the methods for introducing recombinant vectors suited forvarious host cells, which will be described later in the item 3.

Examples of the method for preparing cDNA encoding the enzyme relatingto the synthesis of an intracellular sugar nucleotide, GDP-fucose or theenzyme relating to the modification of a sugar chain wherein 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in the complex N-glycoside-linked sugar chain includethe methods described in the preparation of DNA in the item 1(1)(a) andthe like.

Examples of the method for preparing a genome DNA encoding the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain include the methods in preparation ofgenome DNA described in the item 1(1)(a) and the like.

The nucleotide sequence of the DNA can be determined by digesting itwith appropriate restriction enzymes, cloning the fragments into aplasmid such as pBluescript SK(−) (manufactured by Stratagene) or thelike, subjecting the clones to the reaction generally used as a methodfor analyzing a nucleotide sequence such as the dideoxy method [Proc.Natl. Acad. Sci. USA, 74, 5463 (1977)] of Sanger et al. or the like, andthen analyzing the clones using an automatic nucleotide sequenceanalyzer such as A.L.F. DNA Sequencer (manufactured by Pharmacia) or thelike.

The RDO can be prepared by a usual method or using a DNA synthesizer.

Examples of the method for selecting a cell in which a mutationoccurred, by introducing the ROD into the host cell, in the geneencoding the enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose or the enzyme relating to the modification of asugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain include the methods for directlydetecting mutations in chromosomal genes described in Molecular Cloning,Second Edition, Current Protocols in Molecular Biology and the like;

the methods described in the item 1(1)(a) for selecting a transformantthrough the evaluation of the activity of the introduced enzyme relatingto the synthesis of an intracellular sugar nucleotide, GDP-fucose or theenzyme relating to the modification of a sugar chain wherein 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in the complex N-glycoside-linked sugar chain; themethod for selecting a transformant using the sugar structure of aglycoprotein on the cell membrane which will be described later in theitem 1(5); and the method for selecting a transformant as a measure ofthe sugar structure of the produced antibody molecule which will bedescribed later in the item 5 or 6, and the like.

The construct of the ROD can be designed in accordance with the methodsdescribed in Science, 273, 1386 (1996); Nature Medicine, 4, 285 (1998);Hepatology, 25, 1462 (1997); Gene Therapy, 5, 1960 (1999); J. Mol. Med.,75, 829 (1997); Proc. Natl. Acad. Sci. USA, 96, 8774 (1999); Proc. Natl.Acad. Sci. USA, 96, 8768 (1999); Nuc. Acids. Res., 27, 1323 (1999);Invest. Dematol., 111, 1172 (1998); Nature Biotech., 16, 1343 (1998);Nature Biotech., 18, 43 (2000); Nature Biotech., 18, 555 (2000); and thelike.

(d) Preparation of the Host Cell of the Present Invention by RNAi Method

The host cell of the present invention can be prepared by the RNAi (RNAinterference) method by targeting at a gene of an enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or of anenzyme relating to the modification of a sugar chain wherein 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in the complex N-glycoside-linked sugar chain, forexample, as follows.

A cDNA encoding an enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or an enzyme relating to the modificationof a sugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain is prepared.

The nucleotide sequence of the prepared cDNA is determined.

Based on the determined DNA sequence, an appropriate length of an RNAigene construct comprising the DNA coding moiety encoding the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain or a part of its non-translation region,is designed.

In order to express the RNAi gene in a cell, a recombinant vector isprepared by inserting a fragment or full length of the prepared DNA intodownstream of the promoter of an appropriate expression vector.

A transformant is obtained by introducing the recombinant vector into ahost cell suitable for the expression vector.

The host cell of the present invention can be obtained by selecting atransformant as a measure of the activity of the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or the enzymerelating to the modification of a sugar chain wherein 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex N-glycoside-linked sugar chain, or thesugar chain structure of a glycoprotein on the cell membrane or of theproduced antibody molecule.

As the host cell, any cell such as yeast, animal cell, insect cell orplant cell can be used, so long as it has a gene encoding the targetenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the target enzyme relating to the modification of a sugarchain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain. Examples include the host cells whichwill be described later in the item 3.

As the expression vector, a vector which is autonomously replicable inthe host cell or can be integrated into the chromosome and comprises apromoter at such a position that the designed RNAi gene can betransferred is used. Examples include the expression vectors which willbe described later in the item 3.

As the method for introducing a gene into various host cells, themethods for introducing recombinant vectors suitable for various hostcells, which will be described later in the item 3, can be used.

Examples of the method for selecting a transformant as a measure of theactivity of the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the enzyme relating to the modificationof a sugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain include the methods described in the item1(1)(a).

Examples of the method for selecting a transformant as a measure of thesugar chain structure of a glycoprotein on the cell membrane include themethods which will be described later in the item 1(5). Examples of themethod for selecting a transformant as a measure of the sugar chainstructure of a produced antibody molecule include the methods which willbe described later in the item 5 or 6.

Examples of the method for preparing cDNA encoding the enzyme relatingto the synthesis of an intracellular sugar nucleotide, GDP-fucose or theenzyme relating to the modification of a sugar chain wherein 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in the complex N-glycoside-linked sugar chain includethe methods described in preparation of DNA in the item 1(1)(a) and thelike.

In addition, the host cell of the present invention can also be obtainedwithout using an expression vector, by directly introducing an RNAi genedesigned based on the nucleotide sequence encoding the enzyme relatingto the synthesis of an intracellular sugar nucleotide, GDP-fucose or theenzyme relating to the modification of a sugar chain wherein 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in the complex N-glycoside-linked sugar chain.

The RNAi gene can be prepared in the usual method or using a DNAsynthesizer.

The RNAi gene construct can be designed in accordance with the methodsdescribed in Nature, 391, 806 (1998); Proc. Natl. Acad. Sci. USA, 95,15502 (1998); Nature, 395, 854 (1998); Proc. Natl. Acad. Sci. USA, 96,5049 (1999); Cell, 95, 1017 (1998); Proc. Natl. Acad. Sci. USA, 96, 1451(1999); Proc. Natl. Acad. Sci. USA, 95, 13959 (1998); Nature Cell Biol.,2, 70 (2000); and the like.

(e) Preparation of the Host Cell of the Present Invention by a MethodUsing Transposon

The host cell of the present invention can be prepared by inducingmutation using a transposon system described in Nature Genet., 25, 35(2000) or the like, and then by selecting a mutant as a measure of theactivity of the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the enzyme relating to the modificationof a sugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain, or the sugar chain structure of aglycoprotein of a produced antibody molecule or on the cell membrane.

The transposon system is a system in which a mutation is induced byrandomly inserting an exogenous gene into chromosome, wherein anexogenous gene interposed between transposons is generally used as avector for inducing a mutation, and a transposase expression vector forrandomly inserting the gene into chromosome is introduced into the cellat the same time.

Any transposase can be used, so long as it is suitable for the sequenceof the transposon to be used.

As the exogenous gene, any gene can be used, so long as it can induce amutation in the DNA of a host cell.

As the host cell, any cell such as yeast, animal cell, insect cell orplant cell can be used, so long as it has a gene encoding the targetenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or of the target enzyme relating to the modification of asugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain. Examples include the host cells whichwill be described later in the item 3. For introducing the gene intovarious host cells, the method for introducing recombinant vectorssuitable for various host cells, which will be described later in theitem 3, can be used.

Examples of the method for selecting a mutant as a measure of theactivity of the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the enzyme relating to the modificationof a sugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain include the methods described in the item1(1)(a).

Examples of the method for selecting a mutant as a measure of the sugarchain structure of a glycoprotein on the cell membrane include themethods which will be described later in the item 1(5). Examples of themethod for selecting a mutant as a measure of the sugar chain structureof a produced antibody molecule include the methods which will bedescribed later in the item 5 or 6.

(2) Method for Introducing a Dominant Negative Mutant of a Gene Encodingan Enzyme

The host cell of the present invention can be prepared by targeting agene encoding an enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or an enzyme relating to the modificationof a sugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain, using a technique for introducing adominant negative mutant of the enzyme. Examples of the enzyme relatingto the synthesis of an intracellular sugar nucleotide, GDP-fucoseinclude GMD, Fx, GFPP, fucokinase and the like. Examples of the enzymerelating to the modification of a sugar chain wherein 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex N-glycoside-linked sugar chain includeα-1,6-fucosyltransferase, α-L-fucosidase and the like.

The enzymes catalyze specific reactions having substrate specificity,and dominant negative mutants of the enzymes can be prepared bydisrupting the active center of the enzymes which catalyze the catalyticactivity having substrate specificity. The method for preparing adominant negative mutant is specifically described as follows withreference to GMD among the target enzymes.

As a result of the analysis of the three-dimensional structure of E.coli-derived GMD, it has been revealed that 4 amino acids (threonine atposition 133, glutamic acid at position 135, tyrosine at position 157and lysine at position 161) have an important function on the enzymeactivity (Structure, 8, 2, 2000). That is, when mutants were prepared bysubstituting the 4 amino acids with other different amino acids based onthe three-dimensional structure information, the enzyme activity of allof the mutants was significantly decreased. On the other hand, changesin the ability of GMD to bind to GMD coenzyme NADP and its substrateGDP-mannose were hardly observed in the mutants. Accordingly, a dominantnegative mutant can be prepared by substituting the 4 amino acids whichcontrol the enzyme activity of GMD. For example, in GMD (SEQ ID NO:65)derived from CHO cell, a dominant negative mutant can be prepared bysubstituting threonine position 155, glutamic acid at position 157,tyrosine at position 179 and lysine at position 183 with other aminoacids, by comparing the homology and predicting the three-dimensionalstructure using the amino acid sequence information based on the resultsof the E. coli-derived GMD. Such a gene into which amino acidsubstitution is introduced can be prepared by the site-directedmutagenesis described in Molecular Cloning, Second Edition, CurrentProtocols in Molecular Biology or the like.

The host cell of the present invention can be prepared in accordancewith the method described in Molecular Cloning, Second Edition, CurrentProtocols in Molecular Biology or the like, using the prepared dominantnegative mutant gene of the target enzyme, for example, as follows.

A gene encoding a dominant negative mutant (hereinafter referred to as“dominant negative mutant gene”) of the enzyme relating to the synthesisof an intracellular sugar nucleotide, GDP-fucose or the enzyme relatingto the modification of a sugar chain wherein 1-position of fucose isbound to 6-position of N-acetylglucosamine in the reducing end throughα-bond in the complex N-glycoside-linked sugar chain is prepared.

Based on the prepared full length DNA of dominant negative mutant gene,a DNA fragment of an appropriate length containing a moiety encoding theprotein is prepared, if necessary.

A recombinant vector is produced by inserting the DNA fragment or fulllength DNA into downstream of the promoter of an appropriate expressionvector.

A transformant is obtained by introducing the recombinant vector into ahost cell suitable for the expression vector.

The host cell of the present invention can be prepared by selecting atransformant as a measure of the activity of the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or theactivity of the enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain, or the sugar chain structure of aglycoprotein of a produced antibody molecule or on the cell membrane.

As the host cell, any cell such as yeast, animal cell, insect cell orplant cell can be used, so long as it has a gene encoding the targetenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or of the target enzyme relating to the modification of asugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain. Examples include the host cells whichwill be described later in the item 3.

As the expression vector, a vector which is autonomously replicable inthe host cell or can be integrated into the chromosome and comprises apromoter at a position where transcription of the DNA encoding thedominant negative mutant of interest can be effected is used. Examplesinclude the expression vectors which will be described later in the item3.

For introducing the gene into various host cells, the method forintroducing recombinant vectors suitable for various host cells, whichwill be described later in the item 3, can be used.

Examples of the method for selecting a transformant as a measure of theactivity of the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the enzyme relating to the modificationof a sugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain include the methods described in the item1(1)(a).

Examples of the method for selecting a transformant as a measure of thesugar chain structure of a glycoprotein on the cell membrane include themethods which will be described later in the item 1(5). Examples of themethod for selecting a transformant as a measure of the sugar chainstructure of a produced antibody molecule include the methods which willbe described later in the item 5 or 6.

(3) Method for Introducing a Mutation into an Enzyme

The host cell of the present invention can be prepared by introducing amutation into a gene encoding an enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or an enzyme relating to themodification of a sugar chain wherein 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex N-glycoside-linked sugar chain, and then by selecting a cellline of interest in which the mutation occurred in the enzyme.

Examples of the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose include GMD, Fx, GFPP, fucokinase and thelike. Examples of the enzyme relating to the modification of a sugarchain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain include α-1,6-fucosyltransferase,α-L-fucosidase and the like.

Examples of the method include 1) a method in which a desired cell lineis selected from mutants obtained by a mutation-inducing treatment of aparent cell line with a mutagen or spontaneously generated mutants, as ameasure of the activity of an enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or the activity of an enzymerelating to the modification of a sugar chain wherein 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex N-glycoside-linked sugar chain, 2) amethod in which a desired cell line is selected from mutants obtained bya mutation-inducing treatment of a parent cell line with a mutagen orspontaneously generated mutants, as a measure of the sugar chainstructure of a produced antibody molecule and 3) a method in which adesired cell line is selected from mutants obtained by amutation-inducing treatment of a parent cell line with a mutagen orspontaneously generated mutants, as a measure of the sugar chainstructure of a glycoprotein on the cell membrane.

As the mutation-inducing treatment, any treatment can be used, so longas it can induce a point mutation or a deletion or frame shift mutationin the DNA of cells of the parent cell line.

Examples include treatment with ethyl nitrosourea, nitrosoguanidine,benzopyrene or an acridine pigment and treatment with radiation. Also,various alkylating agents and carcinogens can be used as mutagens.Examples of the method for allowing a mutagen to act upon cells includethe methods described in Tissue Culture Techniques, 3rd edition (AsakuraShoten), edited by Japanese Tissue Culture Association (1996), NatureGenet., 24, 314 (2000) and the like.

Examples of the spontaneously generated mutant include mutants which arespontaneously formed by continuing subculture under general cell cultureconditions without applying special mutation-inducing treatment.

Examples of the method for measuring the activity of the enzyme relatingto the synthesis of an intracellular sugar nucleotide, GDP-fucose or theactivity of the enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain include the methods described in the item1(1)(a). Examples of the method for discriminating the sugar chainstructure of a prepared antibody molecule include the methods which willbe described later in the item 5 or 6. Examples of the method fordiscriminating the sugar chain structure of a glycoprotein on the cellmembrane include the methods which will be described later in the item1(5).

(4) Method for Inhibiting Transcription and/or Translation of a GeneEncoding an Enzyme

The host cell of the present invention can be prepared by inhibitingtranscription and/or translation of a target gene through a method suchas the antisense RNA/DNA technique [Bioscience and Industry, 50, 322(1992); Chemistry, 46, 681 (1991); Biotechnology, 9, 358 (1992); Trendsin Biotechnology, 10, 87 (1992); Trends in Biotechnology, 10, 152(1992); Cell Engineering, 16, 1463 (1997)], the triple helix technique[Trends in Biotechnology, 10, 132 (1992)] or the like, using a geneencoding an enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose and/or an enzyme relating to the modification ofa sugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain, as the target.

Examples of the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose include GMD, Fx, GFPP, fucokinase and thelike. Examples of the enzyme relating to the modification of a sugarchain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain include α-1,6-fucosyltransferase,α-L-fucosidase and the like.

(5) Method for Selecting a Cell Line Resistant to a Lectin whichRecognizes a Sugar Chain Structure in which 1-Position of Fucose isBound to 6-Position of N-Acetylglucosamine in the Reducing End Throughα-Bond in the N-Glycoside-Linked Sugar Chain

The host cell of the present invention can be prepared by using a methodfor selecting a cell line resistant to a lectin which recognizes a sugarchain structure in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in theN-glycoside-linked sugar chain.

Examples of the method for selecting a cell line resistant to a lectinwhich recognizes a sugar chain structure in which 1-position of fucoseis bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the N-glycoside-linked sugar chain include the methodsusing lectin described in Somatic Cell Mol. Genet., 12, 51 (1986) andthe like. As the lectin, any lectin can be used, so long as it is alectin which recognizes a sugar chain structure in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the N-glycoside-linked sugar chain. Examples include aLens culinaris lectin LCA (lentil agglutinin derived from Lensculinaris), a pea lectin PSA (pea lectin derived from Pisum sativum), abroad bean lectin VFA (agglutinin derived from Vicia faba), an Aleuriaaurantia lectin AAL (lectin derived from Aleuria aurantia) and the like.

Specifically, the cell line of the present invention resistant to alectin which recognizes a sugar chain structure in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the N-glycoside-linked sugar chain can be selected byculturing cells for 1 day to 2 weeks, preferably from 1 day to 1 week,using a medium comprising the lectin at a concentration of 1 μg/ml to 1mg/ml, subculturing surviving cells or picking up a colony andtransferring it into a culture vessel, and subsequently continuing theculturing using the lectin-containing medium. Examples of the cell lineobtained by the method include CHO/CCR4-LCA Nega-13 (FERM BP-7756)obtained in Example 14(2) which will be described later.

2. Preparation of a Transgenic Non-Human Animal or Plant or theProgenies Thereof of the Present Invention

The transgenic non-human animal or plant or the progenies thereof of thepresent invention is a transgenic non-human animal or plant or theprogenies thereof in which a genome gene is modified in such a mannerthat the activity of an enzyme relating to the modification of a sugarchain of an antibody molecule can be controlled, and it can be preparedaccording to the method similar to that in the item 1, using a geneencoding an enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose or an enzyme relating to the modification of asugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain, as the target.

In a transgenic non-human animal, the embryonic stem cell of the presentinvention in which the activity of the enzyme relating to the synthesisof an intracellular sugar nucleotide, GDP-fucose or the activity of theenzyme relating to the modification of a sugar chain wherein 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in the complex N-glycoside-linked sugar chain iscontrolled can be prepared applying the method similar to that in theitem 1 to an embryonic stem cell of the intended non-human animal suchas cattle, sheep, goat, pig, horse, mouse, rat, fowl, monkey, rabbit orthe like.

Specifically, a mutant clone is prepared in which a gene encoding theenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain is inactivated or substituted with anysequence, by a known homologous recombination technique [e.g., Nature,326, 6110, 295 (1987); Cell, 51, 3, 503 (1987); or the like]. Using theprepared mutant clone, a chimeric individual comprising an embryonicstem cell clone and a normal cell can be prepared by an injectionchimera method into blastocyst of fertilized egg of an animal or by anaggregation chimera method. The chimeric individual is crossed with anormal individual, so that a transgenic non-human animal in which theactivity of the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the activity of the enzyme relating tothe modification of a sugar chain wherein 1-position of fucose is boundto 6-position of N-acetylglucosamine in the reducing end through α-bondin the complex N-glycoside-linked sugar chain is decreased or deleted inthe whole body cells can be obtained.

Also, a fertilized egg cell of the present invention in which theactivity of an enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the activity of an enzyme relating tothe modification of a sugar chain wherein 1-position of fucose is boundto 6-position of N-acetylglucosamine in the reducing end through α-bondin the complex N-glycoside-linked sugar chain is decreased or deletedcan be prepared by applying the method similar to that in the item 1 tofertilized egg of a non-human animal of interest such as cattle, sheep,goat, pig, horse, mouse, rat, fowl, monkey, rabbit or the like.

A transgenic non-human animal in which the activity of an enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the activity of an enzyme relating to the modification ofa sugar chain wherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain is decreased can be prepared bytransplanting the prepared fertilized egg cell into the oviduct oruterus of a pseudopregnant female using the embryo transplantationmethod described in Manipulating Mouse Embryo, Second Edition or thelike, followed by childbirth by the animal.

In a transgenic plant, the callus of the present invention in which theactivity of an enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the activity of an enzyme relating tothe modification of a sugar chain wherein 1-position of fucose is boundto 6-position of N-acetylglucosamine in the reducing end through α-bondin the complex N-glycoside-linked sugar chain is decreased or deletedcan be prepared by applying the method similar to that in the item 1 toa callus or cell of the plant of interest.

A transgenic plant in which the activity of an enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or theactivity of an enzyme relating to the modification of a sugar chainwherein 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complexN-glycoside-linked sugar chain is decreased can be prepared by culturingthe prepared callus using a medium comprising auxin and cytokinin toredifferentite it in accordance with a known method [Tissue Culture, 20(1994); Tissue Culture, 21 (1995); Trends in Biotechnology, 15, 45(1997)].

3. Method for Producing an Antibody Composition

The antibody composition can be obtained by expressing it in a host cellusing the methods described in Molecular Cloning, Second Edition;Current Protocols in Molecular Biology; Antibodies, A Laboratory Manual,Cold Spring Harbor Laboratory, 1988 (hereinafter referred also to as“Antibodies”); Monoclonal Antibodies: Principles and Practice, ThirdEdition, Acad. Press, 1993 (hereinafter referred also to as “MonoclonalAntibodies”); and Antibody Engineering, A Practical Approach, IRL Pressat Oxford University Press (hereinafter referred also to as “AntibodyEngineering”), for example, as follows.

A full length cDNA encoding an antibody molecule is prepared, and anappropriate length of a DNA fragment comprising a moiety encoding theantibody molecule is prepared.

A recombinant vector is prepared by inserting the DNA fragment or thefull length cDNA into downstream of the promoter of an appropriateexpression vector.

A transformant which produces the antibody molecule can be obtained byintroducing the recombinant vector into a host cell suitable for theexpression vector.

As the host cell, any of yeast, animal cell, insect cell, plant cell orthe like can be used, so long as it can express the gene of interest.

A cell such as yeast, animal cell, insect cell, plant cell or the likeinto which an enzyme relating to the modification of anN-glycoside-linked sugar chain which binds to the Fc region of theantibody molecule is introduced by a genetic engineering technique canalso be used as the host cell.

As the expression vector, a vector which is autonomously replicable inthe host cell or can be integrated into the chromosome and comprises apromoter at such a position that the DNA encoding the antibody moleculeof interest can be transferred is used.

The cDNA can be prepared from a human or non-human tissue or cell using,e.g., a probe primer specific for the antibody molecule of interest, inaccordance with the methods described in the preparation of DNA in theitem 1(1)(a).

When a yeast is used as the host cell, examples of the expression vectorinclude YEP13 (ATCC 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419) andthe like.

Any promoter can be used, so long as it can function in yeast. Examplesinclude a promoter of a gene of the glycolytic pathway such as a hexosekinase gene, etc., PH05 promoter, PGK promoter, GAP promoter, ADHpromoter, gal 1 promoter, gal 10 promoter, heat shock protein promoter,MF α1 promoter, CUP 1 promoter and the like.

Examples of the host cell include microorganisms belonging to the genusSaccharomyces, the genus Schizosaccharomyces, the genus Kluyveromyces,the genus Trichosporon, the genus Schwanniomyces and the like, such asSaccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis, Trichosporon pullulans and Schwanniomyces alluvius, etc.

As the method for introducing the recombinant vector, any method can beused, so long as it can introduce DNA into yeast. Examples includeelectroporation [Methods in Enzymology, 194, 182 (1990)], spheroplastmethod [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium acetatemethod [J. Bacteriol., 153, 163 (1983)], a method described in Proc.Natl. Acad. Sci. USA, 75, 1929 (1978) and the like.

When an animal cell is used as the host, examples of the expressionvector include pcDNAI, pcDM8 (available from Funakoshi), pAGE107[Japanese Published Examined Patent Application No. 22979/91;Cytotechnology, 3, 133 (1990)], pAS3-3 (Japanese Published ExaminedPatent Application No. 227075/90), pCDM8 [Nature, 329, 840 (1987)],pcDNAI/Amp (manufactured by Invitrogen), pREP4 (manufactured byInvitrogen), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210 andthe like.

Any promoter can be used, so long as it can function in an animal cell.Examples include a promoter of IE (immediate early) gene ofcytomegalovirus (CMV), an early promoter of SV40, a promoter ofretrovirus, a promoter of metallothionein, a heat shock promoter, an SRαpromoter and the like. Also, an enhancer of the IE gene of human CMV maybe used together with the promoter.

Examples of the host cell include a human cell such as Namalwa cell, amonkey cell such as COS cell, a Chinese humster cell such as CHO cell orHBT5637 (Japanese Published Examined Patent Application No. 299/88), arat myeloma cell, a mouse myeloma cell, a cell derived from syrianhamster kidney, an embryonic stem cell, a fertilized egg cell and thelike.

As the method for introducing the recombinant vector, any method can beused, so long as it can introduce DNA into an animal cell. Examplesinclude electroporation [Cytotechnology, 3, 133 (1990)], the calciumphosphate method (Japanese Published Examined Patent Application No.227075/90), the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413(1987)], the injection method [Manipulating the Mouse Embryo, ALaboratory Manual], a method using particle gun (gene gun) (JapanesePatent No. 2606856, Japanese Patent No. 2517813), the DEAE-dextranmethod [Biomanual Series 4-Gene Transfer and Expression Analysis(Yodo-sha), edited by Takashi Yokota and Kenichi Arai (1994)], the virusvector method [Manipulating Mouse Embryo, Second Edition] and the like.

When an insect cell is used as the host, the protein can be expressed bythe method described in Current Protocols in Molecular Biology,Baculovirus Expression Vectors, A Laboratory Manual, W.H. Freeman andCompany, New York (1992), Bio/Technology, 6, 47 (1988) or the like.

That is, the protein can be expressed by simultaneously introducing arecombinant gene-introducing vector and a baculovirus into an insectcell to obtain a recombinant virus in an insect cell culture supernatantand then infecting the insect cell with the recombinant virus.

Examples of the gene introducing vector used in the method includepVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and thelike.

Examples of the baculovirus include Autographa californica nuclearpolyhedrosis virus which is infected with an insect of the familyBarathra.

Examples of the insect cell include Spodoptera frugiperda oocytes Sf9and Sf21 [Current Protocols in Molecular Biology, Baculovirus ExpressionVectors, A Laboratory Manual, W.H. Freeman and Company, New York(1992)], a Trichoplusia ni oocyte High 5 (manufactured by Invitrogen)and the like.

Examples of the method for the simultaneously introducing therecombinant gene-introducing vector and the baculovirus for preparingthe recombinant virus include the calcium phosphate method (JapanesePublished Examined Patent Application No. 227075/90), the lipofectionmethod [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like.

When a plant cell is used as the host, examples of the expression vectorinclude Ti plasmid, tobacco mosaic virus and the like.

As the promoter, any promoter can be used, so long as it can function ina plant cell. Examples include cauliflower mosaic virus (CaMV) 35Spromoter, rice actin 1 promoter and the like.

Examples of the host cell include plant cells of tobacco, potato,tomato, carrot, soybean, rape, alfalfa, rice, wheat, barley, etc., andthe like.

As the method for introducing the recombinant vector, any method can beused, so long as it can introduce DNA into a plant cell. Examplesinclude a method using Agrobacterium (Japanese Published Examined PatentApplication No. 140885/84, Japanese Published Examined PatentApplication No. 70080/85, WO 94/00977), electroporation (JapanesePublished Examined Patent Application No. 251887/85), a method using aparticle gun (gene gun) (Japanese Patent No. 2606856, Japanese PatentNo. 2517813) and the like.

As the method for expressing a gene, secretion production, expression ofa fusion protein of the Fc region with other protein and the like can becarried out in accordance with the method described in MolecularCloning, Second Edition or the like, in addition to the directexpression.

When a gene is expressed by a bacterium, a yeast, an animal cell, aninsect cell or a plant cell into which a gene relating to the synthesisof a sugar chain is introduced, an antibody molecule to which a sugar ora sugar chain is added by the introduced gene can be obtained.

An antibody composition can be obtained by culturing the obtainedtransformant in a medium to produce and accumulate the antibody moleculein the culture and then recovering it from the resulting culture. Themethod for culturing the transformant using a medium can be carried outin accordance with a general method which is used for the culturing ofhost cells.

As the medium for culturing a transformant obtained using a prokaryotesuch as Escherichia coli etc. or a eukaryote such as yeast etc. as thehost cell, the medium may be either a natural medium or a syntheticmedium, so long as it comprises materials such as a carbon source, anitrogen source, an inorganic salt and the like which can be assimilatedby the organism and culturing of the transformant can be efficientlycarried out.

As the carbon source, those which can be assimilated by the organism canbe used. Examples include carbohydrates such as glucose, fructose,sucrose, molasses containing them, starch, starch hydrolysate, etc.;organic acids such as acetic acid, propionic acid, etc.; alcohols suchas ethanol, propanol, etc.; and the like.

Examples of the nitrogen source include ammonia; ammonium salts ofinorganic acid or organic acid such as ammonium chloride, ammoniumsulfate, ammonium acetate, ammonium phosphate, etc.; othernitrogen-containing compounds; peptone; meat extract; yeast extract;corn steep liquor; casein hydrolysate; soybean meal; soybean mealhydrolysate; various fermented cells and hydrolysates thereof; and thelike.

Examples of the inorganic material include potassium dihydrogenphosphate, dipotassium hydrogen phosphate, magnesium phosphate,magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate,copper sulfate, calcium carbonate, and the like

The culturing is carried out generally under aerobic conditions such asa shaking culture, submerged-aeration stirring culture or the like. Theculturing temperature is preferably 15 to 40° C., and the culturing timeis generally 16 hours to 7 days. During the culturing, the pH ismaintained at 3.0 to 9.0. The pH is adjusted using an inorganic ororganic acid, an alkali solution, urea, calcium carbonate, ammonia orthe like.

If necessary, an antibiotic such as ampicillin, tetracycline or the likemay be added to the medium during the culturing.

When a microorganism transformed with a recombinant vector obtainedusing an inducible promoter as the promoter is cultured, an inducer maybe added to the medium, if necessary. For example, when a microorganismtransformed with a recombinant vector obtained using lac promoter iscultured, isopropyl-β-D-thiogalactopyranoside may be added to themedium, and when a microorganism transformed with a recombinant vectorobtained using trp promoter is cultured, indoleacrylic acid may be addedto the medium.

When a transformant obtained using an animal cell as the host cell iscultured, examples of the medium include generally used RPMI 1640 medium[The Journal of the American Medical Association, 199, 519 (1967)],Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEMmedium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Societyfor the Biological Medicine, 73, 1 (1950)] and Whitten's medium[Developmental Engineering Experimentation Manual—Preparation ofTransgenic Mice (Kodan-sha), edited by M. Katshuki (1987)], the media towhich fetal calf serum, etc. is added, and the like.

The culturing is carried out generally at a pH of 6 to 8 and 30 to 40°C. for 1 to 7 days in the presence of 5% CO₂. If necessary, anantibiotic such as kanamycin, penicillin or the like may be added to themedium during the culturing.

Examples of the medium for use in the culturing of a transformantobtained using an insect cell as the host include usually used TNM-FHmedium (manufactured by Pharmingen), Sf-900 II SFM medium (manufacturedby Life Technologies), ExCell 400 and ExCell 405 (both manufactured byJRH Biosciences), Grace's Insect Medium [Nature, 195, 788 (1962)] andthe like.

The culturing is carried out generally at a medium pH of 6 to 7 and 25to 30° C. for 1 to 5 days.

In addition, antibiotics such as gentamicin may be added to the mediumduring the culturing as occasion demands.

A transformant obtained using a plant cell as the host can be culturedas a cell or by differentiating it into a plant cell or organ. Examplesof the medium for culturing the transformant include generally usedMurashige and Skoog (MS) medium and White medium, the media to which aplant hormone such as auxin, cytokinin, etc. is added, and the like.

The culturing is carried out generally at a pH of 5 to 9 and 20 to 40°C. for 3 to 60 days.

If necessary, an antibiotic such as kanamycin, hygromycin or the likemay be added to the medium during the culturing.

Accordingly, an antibody composition can be produced by culturing atransformant derived from a microorganism, an animal cell or a plantcell, which comprises a recombinant vector into which a DNA encoding anantibody molecule is inserted, in accordance with a general culturingmethod, to thereby produce and accumulate the antibody composition, andthen recovering the antibody composition from the culture.

As the method for expressing the gene, secretion production, expressionof a fusion protein and the like can be carried out in accordance withthe method described in Molecular Cloning, Second Edition, in additionto the direct expression.

Examples of the method for producing an antibody composition include amethod of intracellular expression in a host cell, a method ofextracellular secretion from a host cell, and a method of production ona host cell membrane outer envelope. The method can be selected bychanging the host cell used or the structure of the antibody compositionproduced.

When the antibody composition of the present invention is produced in ahost cell or on a host cell membrane outer envelope, it can bepositively secreted extracellularly in accordance with the method ofPaulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Loweet al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4,1288 (1990)], the methods described in Japanese Published ExaminedPatent Application No. 336963/93 and Japanese Published Examined PatentApplication No. 823021/94 and the like.

That is, an antibody molecule of interest can be positively secretedextracellularly from a host cell by inserting a DNA encoding theantibody molecule and a DNA encoding a signal peptide suitable for theexpression of the antibody molecule into an expression vector using agene recombination technique, introducing the expression vector into thehost cell and then expressing the antibody molecule.

Also, its production amount can be increased in accordance with themethod described in Japanese Published Examined Patent Application No.227075/90 using a gene amplification system using a dihydrofolatereductase gene.

In addition, the antibody composition can also be produced using agene-introduced animal individual (transgenic non-human animal) or aplant individual (transgenic plant) which is constructed by theredifferentiation of an animal or plant cell into which the gene isintroduced.

When the transformant is an animal individual or a plant individual, anantibody composition can be produced in accordance with a general methodby rearing or cultivating it to thereby produce and accumulate theantibody composition and then recovering the antibody composition fromthe animal or plant individual.

Examples of the method for producing an antibody composition using ananimal individual include a method in which the antibody composition ofinterest is produced in an animal constructed by introducing a gene inaccordance with a known method [American Journal of Clinical Nutrition,63, 627S (1996); Bio/Technology, 9, 830 (1991)].

In the case of an animal individual, an antibody composition can beproduced by rearing a transgenic non-human animal into which a DNAencoding an antibody molecule is introduced to thereby produce andaccumulate the antibody composition in the animal, and then recoveringthe antibody composition from the animal. Examples of the place of theanimal where the composition is produced and accumulated include milk(Japanese Published Examined Patent Application No. 309192/88) and eggsof the animal.

As the promoter used in this case, any promoter can be used, so long asit can function in an animal. Preferred examples include mammary glandcell-specific promoters such as α casein promoter, β casein promoter, βlactoglobulin promoter, whey acidic protein promoter and the like.

Example of the method for producing an antibody composition using aplant individual include a method in which an antibody composition isproduced by cultivating a transgenic plant into which a DNA encoding anantibody molecule is introduced by a known method [Tissue Culture,(1994); Tissue Culture, 21 (1995); Trends in Biotechnology, 15, 45(1997)] to produce and accumulate the antibody composition in the plant,and then recovering the antibody composition from the plant.

Regarding purification of an antibody composition produced by atransformant into which a gene encoding an antibody molecule isintroduced, for example, when the antibody composition isintracellularly expressed in a dissolved state, the cells afterculturing are recovered by centrifugation, suspended in an aqueousbuffer and then disrupted using ultrasonic oscillator, French press,Manton Gaulin homogenizer, dynomill or the like to obtain a cell-freeextract. A purified product of the antibody composition can be obtainedfrom a supernatant obtained by centrifuging the cell-free extract, byusing a general enzyme isolation purification techniques such as solventextraction; salting out; desalting with ammonium sulfate, etc.;precipitation with an organic solvent; anion exchange chromatographyusing a resin such as diethylaminoethyl (DEAE)-Sepharose, DIAION HPA-75(manufactured by Mitsubishi Chemical), etc.; cation exchangechromatography using a resin such as S-Sepharose FF (manufactured byPharmacia), etc.; hydrophobic chromatography using a resin such asbutyl-Sepharose, phenyl-Sepharose, etc.; gel filtration using amolecular sieve; affinity chromatography; chromatofocusing;electrophoresis such as isoelectric focusing, etc.; and the like whichmay be used alone or in combination.

Also, when the antibody composition is expressed intracellularly byforming an insoluble body, the cells are recovered, disrupted andcentrifuged in the same manner, and the insoluble body of the antibodycomposition is recovered as a precipitation fraction. The recoveredinsoluble body of the antibody composition is solubilized using aprotein denaturing agent. The antibody composition is made into a normalthree-dimensional structure by diluting or dialyzing the solubilizedsolution, and then a purified product of the antibody composition isobtained by the same isolation purification method.

When the antibody composition is secreted extracellularly, the antibodycomposition or derivatives thereof can be recovered from the culturesupernatant. That is, the culture is treated by a technique such ascentrifugation or the like to obtain a soluble fraction, and a purifiedpreparation of the antibody composition can be obtained from the solublefraction by the same isolation purification method.

Examples of the thus obtained antibody composition include an antibody,the fragment of the antibody, a fusion protein comprising the Fc regionof the antibody, and the like.

As an example for obtaining the antibody composition, a method forproducing a composition of a humanized antibody is described below indetail, but other antibody compositions can also be obtained in a mannersimilar to the method.

(1) Construction of Vector for Humanized Antibody Expression

A vector for humanized antibody expression is an expression vector foranimal cell into which genes encoding the heavy chain (H chain) andlight chain (L chain) C regions of a human antibody are inserted, whichcan be constructed by cloning each of genes encoding the H chain and Lchain C regions of a human antibody into an expression vector for animalcell.

The C regions of a human antibody may be the H chain and L chain of anyhuman antibody. Examples include the C region belonging to IgG1 subclassin the H chain of a human antibody (hereinafter referred to as “hCγ1”),the C region belonging to κ class in the L chain of a human antibody(hereinafter referred to as “hCκ”), and the like.

As the genes encoding the H chain and L chain C regions of a humanantibody, a chromosomal DNA comprising an exon and an intron can be usedor a cDNA can also be used.

As the expression vector for animal cell, any vector can be used, solong as a gene encoding the C region of a human antibody can be insertedthereinto and expressed therein. Examples include pAGE107[Cytotechnology, 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307(1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci.USA, 78, 1527 (1981), pSG1 β d2-4 [Cytotechnology, 4, 173 (1990)] andthe like. Examples of the promoter and enhancer in the expression vectorfor animal cell include SV40 early promoter and enhancer [J. Biochem.,101, 1307 (1987)], Moloney mouse leukemia virus LTR promoter [Biochem.Biophys. Res. Commun., 149, 960 (1987)], immunoglobulin H chain promoter[Cell, 41, 479 (1985)] and enhancer [Cell, 33, 717 (1983)], and thelike.

The humanized antibody expression vector may be either of a type inwhich genes encoding the H chain and L chain of an antibody exist onseparate vectors or of a type in which both genes exist on the samevector (tandem type). In respect of easiness of construction of ahumanized antibody expression vector, easiness of introduction intoanimal cells, and balance between the expression amounts of the H and Lchains of an antibody in animal cells, a tandem type of the humanizedantibody expression vector is more preferred [J. Immunol. Methods, 167,271 (1994)].

The constructed humanized antibody expression vector can be used forexpression of a human chimeric antibody and a human CDR-grafted antibodyin animal cells.

(2) Preparation of cDNA Encoding V Region of Antibody Derived fromAnimal Other than Human

cDNAs encoding the H chain and L chain V regions of an antibody derivedfrom an animal other than human, such as a mouse antibody, can beobtained in the following manner.

A cDNA is synthesized by extracting mRNA from a hybridoma cell whichproduces the mouse antibody of interest. The synthesized cDNA is clonedinto a vector such as a phage or a plasmid to obtain a cDNA library.Each of a recombinant phage or recombinant plasmid comprising a cDNAencoding the H chain V region and a recombinant phage or recombinantplasmid comprising a cDNA encoding the L chain V region is isolated fromthe library using a C region part or a V region part of an existingmouse antibody as the probe. Full nucleotide sequences of the H chainand L chain V regions of the mouse antibody of interest on therecombinant phage or recombinant plasmid are determined, and full aminoacid sequences of the H chain and L chain V regions are deduced from thenucleotide sequences.

As the animal other than human, any animal such as mouse, rat, hamster,rabbit or the like can be used so long as a hybridoma cell can beproduced therefrom.

Examples of the method for preparing total RNA from a hybridoma cellinclude the guanidine thiocyanate-cesium trifluoroacetate method[Methods in Enzymology, 154, 3 (1987)] and the like, and examples of themethod for preparing mRNA from total RNA, an oligo(dT)-immobilizedcellulose column method (Molecular Cloning, Second Edition) and thelike. In addition, examples of a kit for preparing mRNA from a hybridomacell include Fast Track mRNA Isolation Kit (manufactured by Invitrogen),Quick Prep mRNA Purification Kit (manufactured by Pharmacia) and thelike.

Examples of the method for synthesizing cDNA and preparing a cDNAlibrary include the usual methods (Molecular Cloning, Second Edition,Current Protocols in Molecular Biology, Supplement 1-34), methods usinga commercially available kit such as SuperScript™, Plasmid System forcDNA Synthesis and Plasmid Cloning (manufactured by GIBCO BRL) orZAP-cDNA Synthesis Kit (manufactured by Stratagene), and the like.

In preparing the cDNA library, the vector into which a cDNA synthesizedusing mRNA extracted from a hybridoma cell as the template is insertedmay be any vector so long as the cDNA can be inserted. Examples includeZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK(+) [NucleicAcids Research, 17, 9494 (1989)], λzapII (manufactured by Stratagene),λgt10 and λgt11 [DNA Cloning, A Practical Approach, I, 49 (1985)],Lambda BlueMid (manufactured by Clontech), λExCell, pT7T3 18 U(manufactured by Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)],pUC18 [Gene, 33, 103 (1985)] and the like.

As Escherichia coli into which the cDNA library constructed from a phageor plasmid vector is introduced, any Escherichia coli can be used, solong as the cDNA library can be introduced, expressed and maintained.Examples include XL1-Blue MRF′ [Strategies, 5, 81 (1992)], C600[Genetics, 39, 440 (1954)], Y1088 and Y1090 [Science, 222, 778 (1983)],NM522 [J. Mol. Biol., 166, 1 (1983)], K802 [J. Mol. Biol., 16, 118(1966)], JM105 [Gene, 38, 275 (1985)] and the like.

As the method for selecting a cDNA clone encoding the H chain and Lchain V regions of an antibody derived from an animal other than humanfrom the cDNA library, a colony hybridization or a plaque hybridizationusing an isotope- or fluorescence-labeled probe can be used (MolecularCloning, Second Edition). The cDNA encoding the H chain and L chain Vregions can also be prepared by preparing primers and carrying outpolymerase chain reaction (hereinafter referred to as “PCR”; MolecularCloning, Second Edition; Current Protocols in Molecular Biology,Supplement 1-34) using a cDNA synthesized from mRNA or a cDNA library asthe template.

The nucleotide sequences of the cDNAs can be determined by digesting theselected cDNAs with appropriate restriction enzymes, cloning thefragments into a plasmid such as pBluescript SK(−) (manufactured byStratagene) or the like, carrying out the reaction of a generally usednucleotide sequence analyzing method such as the dideoxy method [Proc.Natl. Acad. Sci. USA, 74, 5463 (1977)] of Sanger et al. or the like andthen analyzing the clones using an automatic nucleotide sequenceanalyzer such as A.L.F. DNA Sequencer (manufactured by Pharmacia) or thelike. Whether or not the obtained cDNAs are encoding the full amino acidsequences of the H chain and L chain V regions of the antibodycontaining a secretory signal sequence can be confirmed by deducing thefull amino acid sequences of the H chain and L chain V regions from thedetermined nucleotide sequence and comparing them with the full aminoacid sequences of the H chain and L chain V regions of known antibodies[Sequences of Proteins of Immunological Interest, US Dep. Health andHuman Services (1991)].

(3) Analysis of Amino Acid Sequence of V Region of Antibody Derived fromAnimal Other than Human

Regarding the full amino acid sequences of the H chain and L chain Vregions of the antibody containing a secretory signal sequence, thelength of the secretory signal sequence and the N-terminal amino acidsequences can be deduced and subgroups to which they belong can also befound, by comparing them with the full amino acid sequences of the Hchain and L chain V regions of known antibodies [Sequences of Proteinsof Immunological Interest, US Dep. Health and Human Services, (1991)].In addition, the amino acid sequences of the H chain and L chain Vregions of each CDR can also be found by comparing them with the aminoacid sequences of the H chain and L chain V regions of known antibodies[Sequences of Proteins of Immunological Interest, US Dep. Health andHuman Services, (1991)].

(4) Construction of Human Chimeric Antibody Expression Vector

A human chimeric antibody expression vector can be constructed bycloning cDNAs encoding the H chain and L chain V regions of an antibodyderived from an animal other than human into upstream of genes encodingthe H chain and L chain C regions of a human antibody in the vector forhumanized antibody expression constructed in the item 3(1). For example,a human chimeric antibody expression vector can be constructed bylinking each of cDNAs encoding the H chain and L chain V regions of anantibody derived from an animal other than human to a synthetic DNAcomprising nucleotide sequences at the 3′-terminals of the H chain and Lchain V regions of an antibody derived from an animal other than humanand nucleotide sequences at the 5′-terminals of the H chain and L chainC regions of a human antibody and also having a recognition sequence ofan appropriate restriction enzyme at both terminals, and by cloning theminto upstream of genes encoding the H chain and L chain C regions of ahuman antibody contained in the vector for humanized antibody expressionconstructed described in the item 3(1).

(5) Construction of cDNA Encoding V Region of Human CDR-Grafted Antibody

cDNAs encoding the H chain and L chain V regions of a human CDR-graftedantibody can be obtained as follows. First, amino acid sequences of theframeworks (hereinafter referred to as “FR”) of the H chain and L chainV regions of a human antibody for grafting CDR of the H chain and Lchain V regions of an antibody derived from an animal other than humanis selected. As the amino acid sequences of FRs of the H chain and Lchain V regions of a human antibody, any amino acid sequences can beused so long as they are derived from a human antibody. Examples includeamino acid sequences of FRs of the H chain and L chain V regions ofhuman antibodies registered at databases such as Protein Data Bank,etc., amino acid sequences common in each subgroup of FRs of the H chainand L chain V regions of human antibodies [Sequences of Proteins ofImmunological Interest, US Dep. Health and Human Services (1991)] andthe like. But in order to produce a human CDR-grafted antibody havingpotent activity, it is preferable to select an amino acid sequencehaving a homology as high as possible (at least 60% or more) with aminoacid sequences of the H chain and L chain V regions of an antibody ofinterest derived from an animal other than human.

Next, the amino acid sequences of CDRs of the H chain and L chain Vregions of the antibody of interest derived from an animal other thanhuman are grafted to the selected amino acid sequences of FRs of the Hchain and L chain V regions of a human antibody to design amino acidsequences of the H chain and L chain V regions of the human CDR-graftedantibody. The designed amino acid sequences are converted into DNAsequences by considering the frequency of codon usage found innucleotide sequences of antibody genes [Sequences of Proteins ofImmunological Interest, US Dep. Health and Human Services (1991)], andthe DNA sequences encoding the amino acid sequences of the H chain and Lchain V regions of the human CDR-grafted antibody are designed. Based onthe designed DNA sequences, several synthetic DNA fragments having alength of about 100 bases are synthesized, and PCR is carried out usingthem. In this case, it is preferable in each of the H chain and the Lchain that 6 synthetic DNAs are designed in view of the reactionefficiency of PCR and the lengths of DNAs which can be synthesized.

Also, they can be easily cloned into the vector for humanized antibodyexpression constructed in the item 3(1) by introducing recognitionsequences of an appropriate restriction enzyme into the 5′-terminals ofthe synthetic DNA present on both terminals. After the PCR, theamplified product is cloned into a plasmid such as pBluescript SK(−)(manufactured by Stratagene) or the like and the nucleotide sequencesare determined by the method in the item 3(2) to thereby obtain aplasmid having DNA sequences encoding the amino acid sequences of the Hchain and L chain V regions of the desired human CDR-grafted antibody.

(6) Construction of Human CDR-Grafted Antibody Expression Vector

A human CDR-grafted antibody expression vector can be constructed bycloning the cDNAs encoding the H chain and L chain V regions of thehuman CDR-grafted antibody constructed in the item 3(5) into upstream ofthe gene encoding H chain and L chain C regions of a human antibody inthe vector for humanized antibody expression described in the item 3(1).For example, the human CDR-grafted antibody expression vector can beconstructed by introducing recognizing sequences of an appropriaterestriction enzyme into the 5′-terminals of both terminals of asynthetic DNA fragment, among the synthetic DNA fragments which are usedwhen PCR is carried out in the item 3(5) for constructing the H chainand L chain V regions of the human CDR-grafted antibody, so that theyare cloned into upstream of the genes encoding the H chain and L chain Cregions of a human antibody in the vector for humanized antibodyexpression described in the item 3(1) in such a manner that they can beexpressed in a suitable form.

(7) Stable Production of Humanized Antibody

A transformant capable of stably producing a human chimeric antibody anda human CDR-grafted antibody (both hereinafter referred to as “humanizedantibody”) can be obtained by introducing the humanized antibodyexpression vectors described in the items 3(4) and (6) into anappropriate animal cell.

Examples of the method for introducing a humanized antibody expressionvector into an animal cell include electroporation [Japanese PublishedExamined Patent Application No. 257891/90, Cytotechnology, 3, 133(1990)] and the like.

As the animal cell into which a humanized antibody expression vector isintroduced, any cell can be used so long as it is an animal cell whichcan produce the humanized antibody.

Examples include mouse myeloma cells such as NSO cell and SP2/0 cell,Chinese hamster ovary cells such as CHO/dhfr⁻ cell and CHO/DG44 cell,rat myeloma such as YB2/0 cell and IR983F cell, BHK cell derived from asyrian hamster kidney, a human myeloma cell such as Namalwa cell, andthe like, and a Chinese hamster ovary cell CHO/DG44 cell, a rat myelomaYB2/0 cell and the host cells of the present invention described in theitem 5 are preferred.

After introduction of the humanized antibody expression vector, atransformant capable of stably producing the humanized antibody can beselected using a medium for animal cell culture comprising an agent suchas G418 sulfate (hereinafter referred to as “G418”; manufactured bySIGMA) and the like in accordance with the method disclosed in JapanesePublished Examined Patent Application No. 257891/90. Examples of themedium for animal cell culture include RPMI 1640 medium (manufactured byNissui Pharmaceutical), GIT medium (manufactured by NihonPharmaceutical), EX-CELL 302 medium (manufactured by JRH), IMDM medium(manufactured by GIBCO BRL), Hybridoma-SFM medium (manufactured by GIBCOBRL) media obtained by adding various additives such as fetal bovineserum (hereinafter referred to as “FBS”) to these media, and the like.The humanized antibody can be produced and accumulated in the culturesupernatant by culturing the obtained transformant in a medium. Theexpression level and antigen binding activity of the humanized antibodyin the culture supernatant can be measured by a method such asenzyme-linked immunosorbent assay [hereinafter referred to as “ELISA”;Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter14 (1998), Monoclonal Antibodies: Principles and Practice, AcademicPress Limited (1996)] or the like. Also, the expression level of thehumanized antibody by the transformant can be increased using a DHFRgene amplification system in accordance with the method disclosed inJapanese Published Examined Patent Application No. 257891/90.

The humanized antibody can be purified from a culture supernatant of thetransformant using a protein A column [Antibodies, A Laboratory Manual,Cold Spring Harbor Laboratory, Chapter 8 (1988), Monoclonal Antibodies:Principles and Practice, Academic Press Limited (1996)]. In addition,purification methods generally used for the purification of proteins canalso be used. For example, the purification can be carried out throughthe combination of a gel filtration, an ion exchange chromatography andan ultrafiltration. The molecular weight of the H chain, L chain orantibody molecule as a whole of the purified humanized antibody can bemeasured, e.g., by polyacrylamide gel electrophoresis [hereinafterreferred to as “SDS-PAGE”; Nature, 227, 680 (1970)], Western blotting[Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter12, (1988), Monoclonal Antibodies: Principles and Practice, AcademicPress Limited (1996)] or the like.

Thus, methods for producing an antibody composition using an animal cellas the host have been described, but, as described above, the antibodycomposition can also be produced by a yeast, an insect cell, a plantcell, an animal individual or a plant individual by the same methods onthe animal cell.

When a host cell has the ability to express an antibody moleculeinnately, the antibody composition of the present invention can beproduced by preparing a cell expressing an antibody molecule using themethod described in the item 1, culturing the cell and then purifyingthe antibody composition of interest from the resulting culture.

4. Activity Evaluation of Antibody Composition

As the method for measuring the amount of the purified antibodycomposition, the activity to bind to an antibody and the effectorfunction of the purified antibody composition, the known methoddescribed in Monoclonal Antibodies, Antibody Engineering and the likecan be used.

As the examples, when the antibody composition is a humanized antibody,the binding activity with an antigen and the binding activity with anantigen-positive cultured cell line can be measured by methods such asELISA, an immunofluorescent method [Cancer Immunol. Immunother., 36, 373(1993)] and the like. The cytotoxic activity against an antigen-positivecultured cell line can be evaluated by measuring CDC activity, ADCCactivity [Cancer Immunol. Immunother., 36, 373 (1993)] and the like.

Also, safety and therapeutic effect of the antibody composition in humancan be evaluated using an appropriate model of animal species relativelyclose to human, such as Macaca fascicularis or the like.

5. Analysis of Sugar Chains Binding to Antibody Molecules Expressed inVarious Cells

The sugar chain structure binding to an antibody molecule expressed invarious cells can be analyzed in accordance with the general analysis ofthe sugar chain structure of a glycoprotein. For example, the sugarchain which is bound to IgG molecule comprises a neutral sugar such asgalactose, mannose, fucose or the like, an amino sugar such asN-acetylglucosamine or the like and an acidic sugar such as sialic acidor the like, and can be analyzed by a method such as a sugar chainstructure analysis or the like using sugar composition analysis, twodimensional sugar chain mapping or the like.

(1) Analysis of Neutral Sugar and Amino Sugar Compositions

The sugar chain composition binding to an antibody molecule can beanalyzed by carrying out acid hydrolysis of sugar chains with an acidsuch as trifluoroacetic acid or the like to release a neutral sugar oran amino sugar and measuring the composition ratio.

Example include a method using a sugar composition analyzer (BioLC)manufactured by Dionex. The BioLC is an apparatus which analyzes a sugarcomposition by HPAEC-PAD (high performance anion-exchangechromatography-pulsed amperometric detection) [J. Liq. Chromatogr., 6,1577 (1983)].

The composition ratio can also be analyzed by a fluorescence labelingmethod using 2-aminopyridine. Specifically, the compositional ratio canbe calculated in accordance with a known method [Agric. Biol. Chem.,55(1), 283-284 (1991)], by labeling an acid-hydrolyzed sample with afluorescence with 2-aminopyridylation and then analyzing the compositionby HPLC.

(2) Analysis of Sugar Chain Structure

The sugar chain structure binding to an antibody molecule can beanalyzed by the two dimensional sugar chain mapping method [Anal.Biochem., 171, 73 (1988), Biochemical Experimentation Methods 23—Methodsfor Studying Glycoprotein Sugar Chains (Japan Scientific SocietiesPress) edited by Reiko Takahashi (1989)]. The two dimensional sugarchain mapping method is a method for deducing a sugar chain structureby, e.g., plotting the retention time or elution position of a sugarchain by reverse phase chromatography as the X axis and the retentiontime or elution position of the sugar chain by normal phasechromatography as the Y axis, respectively, and comparing them with suchresults of known sugar chains.

Specifically, sugar chains are released from an antibody by subjectingthe antibody to hydrazinolysis, and the released sugar chain issubjected to fluorescence labeling with 2-aminopyridine (hereinafterreferred to as “PA”) [J. Biochem., 95, 197 (1984)], and then the sugarchains are separated from an excess PA-treating reagent by gelfiltration, and subjected to reverse phase chromatography. Thereafter,each peak of the separated sugar chains are subjected to normal phasechromatography. The sugar chain structure can be deduced by plotting theresults on a two dimensional sugar chain map and comparing them with thespots of a sugar chain standard (manufactured by Takara Shuzo) or aliterature [Anal. Biochem., 171, 73 (1988)].

The structure deduced by the two dimensional sugar chain mapping methodcan be confirmed by further carrying out mass spectrometry such asMALDI-TOF-MS of each sugar chain or the like.

6. Immunological Determination Method for Discriminating Sugar ChainStructure of Antibody Molecule

An antibody composition comprises an antibody molecule in which sugarchains binding to the Fc region of the antibody are different instructure. The antibody composition in which the ratio of a sugar chainin which fucose is not bound to N-acetylglucosamine in the reducing endin the sugar chain is 20% or more among the total complexN-glycoside-linked sugar chains binding to the Fc region in the antibodycomposition reducing end has potent ADCC activity. The antibodycomposition can be identified by using the method for analyzing thesugar chain structure of an antibody molecule described in the item 6.Also, it can also be identified by an Immunological determination methodusing a lectin.

The sugar chain structure of an antibody molecule can be identified bythe Immunological determination method using a lectin in accordance withthe known Immunological determination method such as Western staining,IRA (radioimmunoassay), VIA (viroimmunoassay), EIA (enzymoimmunoassay),FIA (fluoroimmunoassay), MIA (metalloimmunoassay) and the like describedin Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc.(1995); Immunoassay, 3rd Ed., Igakushoin (1987); Enzyme Antibody Method,Revised Edition, Gakusai Kikaku (1985); and the like.

A lectin which recognizes the sugar chain structure of an antibodymolecule comprised in an antibody composition is labeled, and thelabeled lectin is allowed to react with an antibody composition which isa sample. Then, the amount of the complex of the labeled lectin with theantibody molecule is measured.

Examples of the lectin used for identifying the sugar chain structure ofan antibody molecule include WGA (wheat-germ agglutinin derived from T.vulgaris), ConA (cocanavalin A derived from C. ensiformis), RIC (a toxinderived from R. communis), L-PHA (leucoagglutinin derived from P.vulgaris), LCA (lentil agglutinin derived from L. culinaris), PSA (pealectin derived from P. sativum), AAL (Aleuria aurantia lectin), ACL(Amaranthus caudatus lectin), BPL (Bauhinia purpurea lectin), DSL(Datura stramonium lectin), DBA (Dolichos biflorus agglutinin), EBL(elderberry balk lectin), ECL (Erythrina cristagalli lectin), EEL(Euonymus eoropaeus lectin), GNL (Galanthus nivalis lectin), GSL(Griffonia simplicifolia lectin), HPA (Helix pomatia agglutinin), HHL(Hippeastrum hybrid lectin), Jacalin, LTL (Lotus tetragonolobus lectin),LEL (Lycopersicon esculentum lectin), MAL (Maackia amurensis lectin),MPL (Maclura pomifera lectin), NPL (Narcissus pseudonarcissus lectin),PNA (peanut agglutinin), E-PHA (Phaseolus vulgaris erythroagglutinin),PTL (Psophocarpus tetragonolobus lectin), RCA (Ricinus communisagglutinin), STL (Solanum tuberosum lectin), SJA (Sophora japonicaagglutinin), SBA (soybean agglutinin), UEA (Ulex europaeus agglutinin),VVL (Vicia villosa lectin) and WFA (Wisteria floribunda agglutinin).

It is preferable to use a lectin which specifically recognizes a sugarchain structure wherein fucose binds to the N-acetylglucosamine in thereducing end in the complex N-glycoside-linked sugar chain. Examplesinclude Lens culinaris lectin LCA (lentil agglutinin derived from Lensculinaris), pea lectin PSA (pea lectin derived from Pisum sativum),broad bean lectin VFA (agglutinin derived from Vicia faba) and Aleuriaaurantia lectin AAL (lectin derived from Aleuria aurantia).

7. Application of Antibody Molecule of the Present Invention

The antibody composition of the present invention has potentantibody-dependent cell-mediated cytotoxic activity. An antibody havingpotent antibody-dependent cell-mediated cytotoxic activity is useful forpreventing and treating various diseases including cancers, inflammatorydiseases, immune diseases such as autoimmune diseases, allergies and thelike, circulatory organ diseases and viral or bacterial infections.

In the case of cancers, namely malignant tumors, cancer cells grow.General anti-tumor agents inhibit the growth of cancer cells. Incontrast, an antibody having potent antibody-dependent cell-mediatedcytotoxic activity can treat cancers by injuring cancer cells throughits cell killing effect, and therefore, it is more effective as atherapeutic agent than the general anti-tumor agents. At present, in thetherapeutic agent for cancers, an anti-tumor effect of an antibodymedicament alone is insufficient so that combination therapy withchemotherapy has been carried out [Science, 280, 1197 (1998)]. If morepotent anti-tumor effect is found by the antibody composition of thepresent invention alone, the dependency on chemotherapy will bedecreased and side effects will be reduced.

In immune diseases such as inflammatory diseases, autoimmune diseases,allergies and the like, in vivo reactions of the diseases are induced bythe release of a mediator molecule by immunocytes, so that the allergyreaction can be inhibited by eliminating immunocytes using an antibodyhaving potent antibody-dependent cell-mediated cytotoxic activity.

Examples of the circulatory organ diseases include arteriosclerosis andthe like. The arteriosclerosis is treated using balloon catheter atpresent, but circulatory organ diseases can be prevented and treated byinhibiting growth of arterial cells in restricture after treatment usingan antibody having potent antibody-dependent cell-mediated cytotoxicactivity.

Various diseases including viral and bacterial infections can beprevented and treated by inhibiting proliferation of cells infected witha virus or bacterium using an antibody having potent antibody-dependentcell-mediated cytotoxic activity.

Examples of an antibody which recognizes a tumor-related antigen, anantibody which recognizes an allergy- or inflammation-related antigen,an antibody which recognizes circulatory organ disease-related antigenand an antibody which recognizes a viral or bacterial infection-relatedantigen are described below.

Examples of the antibody which recognizes a tumor-related antigeninclude anti-GD2 antibody (Ohta et al., Anticancer Res., 13, 331-336,1993), anti-GD3 antibody (Ohta et al., Cancer Immunol. Immunother., 36,260-266, 1993), anti-GM2 antibody (Nakamura et al., Cancer Res., 54,1511-1516, 1994), anti-HER2 antibody (Carter et al., Proc. Natl. Acad.Sci. USA, 89, 4285-4289, 1992), anti-CD52 antibody (Carter et al., Proc.Natl. Acad. Sci. USA, 89, 4285-4289, 1992), anti-MAGE antibody(Jungbluth et al., British J. Cancer, 83, 493-497, 2000), anti-HM1.24antibody (Ono et al., Molecular Immunol., 36, 387-395, 1999),anti-parathyroid hormone-related protein (PTHrP) antibody (Ogata et al.,Cancer, 88, 2909-2911, 2000), anti-basic fibroblast growth factorantibody and anti-FGF8 antibody (Matsuzaki et al., Proc. Natl. Acad.Sci. USA, 86, 9911-9915, 1989), anti-basic fibroblast growth factorreceptor antibody and anti-FGF8 receptor antibody (Kuo et al., J. Biol.Chem., 265, 16455-16463, 1990), anti-insulin-like growth factor antibody(Yao et al., J. Neurosci. Res., 40, 647-659, 1995), anti-insulin-likegrowth factor receptor antibody (Yao et al., J. Neurosci. Res., 40,647-659, 1995), anti-PMSA antibody (Murphy et al., J. Urology, 160,2396-2401, 1998), anti-vascular endothelial cell growth factor antibody(Presta et al., Cancer Res., 57, 4593-4599, 1997), anti-vascularendothelial cell growth factor receptor antibody (Kanno et al.,Oncogene, 19, 2138-2146, 2000) and the like.

Examples of the antibody which recognizes an allergy- orinflammation-related antigen include anti-interleukin 6 antibody (Abramset al., Immunol. Rev., 127, 5-24, 1992), anti-interleukin 6 receptorantibody (Sato et al., Molecular Immunol., 31, 371-381, 1994),anti-interleukin 5 antibody (Abrams et al., Immunol. Rev., 127, 5-24,1992), anti-interleukin 5 receptor antibody and anti-interleukin 4antibody (Biord et al., Cytokine, 3, 562-567, 1991), anti-tumor necrosisfactor antibody (Tempest et al., Hybridoma, 13, 183-190, 1994),anti-tumor necrosis factor receptor antibody (Amrani et al., MolecularPharmacol., 58, 237-245, 2000), anti-CCR4 antibody (Campbell et al.,Nature, 400, 776-780, 1999), anti-chemokine antibody (Peri et al., J.Immuno. Meth., 174, 249-257, 1994), anti-chemokine receptor antibody (Wuet al., J. Exp. Med., 186, 1373-1381, 1997) and the like. Examples ofthe antibody which recognizes a circulatory organ disease-relatedantigen include anti-GpIIb/IIIa antibody (Co et al., J. Immunol., 152,2968-2976, 1994), anti-platelet-derived growth factor antibody (Ferns etal., Science, 253, 1129-1132, 1991), anti-platelet-derived growth factorreceptor antibody (Shulman et al., J. Biol. Chem., 272, 17400-17404,1997) and anti-blood coagulation factor antibody (Peter et al.,Circulation, 101, 1158-1164, 2000) and the like.

Examples of the antibody which recognizes a viral or bacterialinfection-related antigen include anti-gp120 antibody (Tugarinov et al.,Structure, 8, 385-395, 2000), anti-CD4 antibody (Schulze-Koops et al.,J. Rheumatology, 25, 2065-2076, 1998), anti-CCR4 antibody and anti-Verotoxin antibody (Karnali et al., J. Clin. Microbiol., 37, 396-399, 1999)and the like.

These antibodies can be obtained from public organizations such as ATCC(The American Type Culture Collection), RIKEN Gene Bank at The Instituteof Physical and Chemical Research, National Institute of Bioscience andHuman Technology, Agency of Industrial Science and Technology (presentname, International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology) and the like, or privatereagent sales companies such as Dainippon Pharmaceutical, R & D SYSTEMS,PharMingen, Cosmo Bio, Funakoshi and the like.

The medicament comprising the antibody composition of the presentinvention can be administered as a therapeutic agent alone, butgenerally, it is preferable to provide it as a pharmaceuticalformulation produced by an appropriate method well known in thetechnical field of manufacturing pharmacy, by mixing it with at leastone pharmaceutically acceptable carrier.

It is desirable to select a route of administration which is mosteffective in treatment. Examples include oral administration andparenteral administration, such as buccal, tracheal, rectal,subcutaneous, intramuscular, intravenous or the like. In an antibodypreparation, intravenous administration is preferable.

The dosage form includes sprays, capsules, tablets, granules, syrups,emulsions, suppositories, injections, ointments, tapes and the like.

Examples of the pharmaceutical preparation suitable for oraladministration include emulsions, syrups, capsules, tablets, powders,granules and the like.

Liquid preparations, such as emulsions and syrups, can be producedusing, as additives, water; saccharides, such as sucrose, sorbitol,fructose, etc.; glycols, such as polyethylene glycol, propylene glycol,etc.; oils, such as sesame oil, olive oil, soybean oil, etc.;antiseptics, such as p-hydroxybenzoic acid esters, etc.; flavors, suchas strawberry flavor, peppermint, etc.; and the like.

Capsules, tablets, powders, granules and the like can be produced using,as additive, fillers, such as lactose, glucose, sucrose, mannitol, etc.;disintegrating agents, such as starch, sodium alginate, etc.;lubricants, such as magnesium stearate, talc, etc.; binders, such aspolyvinyl alcohol, hydroxypropylcellulose, gelatin, etc.; surfactants,such as fatty acid ester, etc.; plasticizers, such as glycerine, etc.;and the like.

Examples of the pharmaceutical preparation suitable for parenteraladministration include injections, suppositories, sprays and the like.

Injections may be prepared using a carrier, such as a salt solution, aglucose solution, a mixture of both thereof or the like. Also, powderedinjections can be prepared by freeze-drying the antibody composition inthe usual way and adding sodium chloride thereto.

Suppositories may be prepared using a carrier such as cacao butter,hydrogenated fat, carboxylic acid or the like.

Also, sprays may be prepared using the antibody composition as such orusing a carrier which does not stimulate the buccal or airway mucousmembrane of the patient and can facilitate absorption of the antibodycomposition by dispersing it as fine particles.

Examples of the carrier include lactose, glycerol and the like.Depending on the properties of the antibody composition and the carrier,it is possible to produce pharmaceutical preparations such as aerosols,dry powders and the like. In addition, the components exemplified asadditives for oral preparations can also be added to the parenteralpreparations.

Although the clinical dose or the frequency of administration variesdepending on the objective therapeutic effect, administration method,treating period, age, body weight and the like, it is usually 10 μg/kgto 20 mg/kg per day and per adult.

Also, as the method for examining antitumor effect of the antibodycomposition against various tumor cells, in vitro tests include CDCactivity measuring method, ADCC activity measuring method and the like,and in vivo tests include antitumor experiments using a tumor system inan experimental animal such as a mouse, etc. and the like.

CDC activity and ADCC activity measurements and antitumor experimentscan be carried out in accordance with the methods described in CancerImmunology Immunotherapy, 36, 373 (1993); Cancer Research, 54, 1511(1994) and the like.

The present invention will be described below in detail based onExamples; however, Examples are only simple illustrations, and the scopeof the present invention is not limited thereto.

Example 1 Production of Anti-Ganglioside GD3 Human Chimeric Antibody

1. Construction of Tandem Expression Vector pChiLHGM4 forAnti-Ganglioside GD3 Human Chimeric Antibody

A plasmid pChi641LGM40 was constructed by ligating a fragment of about4.03 kb containing an L chain cDNA, obtained by digesting an L chainexpression vector, pChi641LGM4 [J. Immunol. Methods, 167, 271 (1994)]for anti-ganglioside GD3 human chimeric antibody (hereinafter referredto as “anti-GD3 chimeric antibody”) with restriction enzymes MluI(manufactured by Takara Shuzo) and SalI (manufactured by Takara Shuzo)with a fragment of about 3.40 kb containing a G418-resistant gene and asplicing signal, obtained by digesting an expression vector pAGE107[Cytotechnology, 3, 133 (1990)] for animal cell with restriction enzymesMluI (manufactured by Takara Shuzo) and SalI (manufactured by TakaraShuzo) using DNA Ligation Kit (manufactured by Takara Shuzo), and thentransforming E. coli HB101 (Molecular Cloning, Second Edition) with theligated product.

Next, a fragment of about 5.68 kb containing an L chain cDNA, obtainedby digesting the constructed plasmid pChi641LGM40 with a restrictionenzyme ClaI (manufactured by Takara Shuzo), blunt-terminating it usingDNA Blunting Kit (manufactured by Takara Shuzo) and further digesting itwith MluI (manufactured by Takara Shuzo), was ligated with a fragment ofabout 8.40 kb containing an H chain cDNA, obtained by digesting ananti-GD3 chimeric antibody H chain expression vector, pChi641HGM4 [J.Immunol. Methods, 167, 271 (1994)] with a restriction enzyme, XhoI(manufactured by Takara Shuzo), blunt-terminating it using DNA BluntingKit (manufactured by Takara Shuzo) and further digesting it with MluI(manufactured by Takara Shuzo), using DNA Ligation Kit (manufactured byTakara Shuzo), and then E. coli HB101 (Molecular Cloning, SecondEdition) was transformed with the ligated product to thereby construct atandem expression vector pChi641LHGM4 for anti-GD3 chimeric antibody.

2. Preparation of Cells Stably Producing Anti-GD3 Chimeric Antibody

Cells capable of stably producing an anti-GD3 chimeric antibody wereprepared using the tandem expression vector pChi641LHGM4 for anti-GD3chimeric antibody constructed in the item 1 of Example 1, as describedbelow.

(1) Preparation of Antibody-Producing Cell Using Rat Myeloma YB2/0 Cell

After introducing 5 μg of the anti-GD3 chimeric antibody expressionvector pChi641LHGM4 into 4×10⁶ cells of rat myeloma YB2/0 [ATCCCRL-1662, J. V. Kilmarin et al., J. Cell. Biol., 93, 576-582 (1982)] byelectroporation [Cytotechnology, 3, 133 (1990)], the cells weresuspended in 40 ml of RPMI1640-FBS(10) (RPMI1640 medium comprising 10%FBS (manufactured by GIBCO BRL)) and dispensed in 200 μl/well into a 96well culture plate (manufactured by Sumitomo Bakelite). After culturingthem at 37° C. for 24 hours in a 5% CO₂ incubator, G418 was added to aconcentration of 0.5 mg/ml, followed by culturing for 1 to 2 weeks. Theculture supernatant was recovered from wells in which colonies oftransformants showing G418 resistance were formed and growth of colonieswas observed, and the antigen binding activity of the anti-GD3 chimericantibody in the supernatant was measured by the ELISA shown in the item3 of Example 1.

Regarding the transformants in wells in which production of the anti-GD3chimeric antibody was observed in culture supernatants, in order toincrease the amount of the antibody production using a DHFR geneamplification system, each of them was suspended in the RPMI1640-FBS(10)medium comprising 0.5 mg/ml G418 and 50 nM DHFR inhibitor, methotrexate(hereinafter referred to as “MTX”; manufactured by SIGMA) to give adensity of 1 to 2×10⁵ cells/ml, and the suspension was dispensed in 2 mlinto wells of a 24 well plate (manufactured by Greiner). Transformantsshowing 50 nM MTX resistance were induced by culturing at 37° C. for 1to 2 weeks in a 5% CO₂ incubator. The antigen binding activity of theanti-GD3 chimeric antibody in culture supernatants in wells in whichgrowth of transformants was observed was measured by the ELISA shown inthe item 3 of Example 1. Regarding the transformants in wells in whichproduction of the anti-GD3 chimeric antibody was observed in culturesupernatants, the MTX concentration was increased to 100 nM and then to200 nM, and transformants capable of growing in the RPMI1640-FBS(10)medium comprising 0.5 mg/ml G418 and 200 nM MTX and of producing theanti-GD3 chimeric antibody in a large amount were finally obtained bythe same method as described above. Among the obtained transformants,suitable cell lines were selected and were made into a single cell(cloning) by limiting dilution twice. Also, using the method fordetermining the transcription product of an α-1,6-fucosyltransferasegene shown in Example 9, a cell line producing a relatively small amountof the transcription product was selected and used as a suitable cellline.

The obtained anti-GD3 chimeric antibody-producing transformed cell clone7-9-51 has been deposited on Apr. 5, 1999, as FERM BP-6691 in NationalInstitute of Bioscience and Human Technology, Agency of IndustrialScience and Technology (Higashi 1-1-3, Tsukuba, Ibaraki, Japan) (presentname: International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1,Higashi 1-Chome Tsukuba-shi, Ibaraki-ken, Japan)).

(2) Preparation of Antibody-Producing Cell Using CHO/DG44 cell

After introducing 4 μg of the anti-GD3 chimeric antibody expressionvector, pChi641LHGM4, into 1.6×10⁶ cells of CHO/DG44 [G. Urlaub and L.A. Chasin, Proc. Natl. Acad. Sci. USA, 77, 4216-4220 (1980)] byelectroporation [Cytotechnology, 3, 133 (1990)], the cells weresuspended in 10 ml of IMDM-FBS(10) [IMDM medium comprising 10% FBS and1× concentration of HT supplement (manufactured by GIBCO BRL)] anddispensed in 200 μl/well into a 96 well culture plate (manufactured byIwaki Glass). After culturing them at 37° C. for 24 hours in a 5% CO₂incubator, G418 was added to give a concentration of 0.5 mg/ml, followedby culturing for 1 to 2 weeks. The culture supernatant was recoveredfrom wells in which colonies of transformants showing G418 resistancewere formed and growth of colonies was observed, and the antigen bindingactivity of the anti-GD3 chimeric antibody in the supernatant wasmeasured by the ELISA shown in the item 3 of Example 1.

Regarding the transformants in wells in which production of the anti-GD3chimeric antibody was observed in culture supernatants, in order toincrease the amount of the antibody production using a DHFR geneamplification system, each of them was suspended in an IMDM-dFBS(10)medium [IMDM medium comprising 10% dialyzed fetal bovine serum(hereinafter referred to as “dFBS”; manufactured by GIBCO BRL)]comprising 0.5 mg/ml G418 and 10 nM MTX to give a density of 1 to 2×10⁵cells/ml, and the suspension was dispensed in 0.5 ml into wells of a 24well plate (manufactured by Iwaki Glass). Transformants showing 10 nMMTX resistance were induced by culturing at 37° C. for 1 to 2 weeks in a5% CO₂ incubator. Regarding the transformants in wells in which theirgrowth was observed, the MTX concentration was increased to 100 nM, andtransformants capable of growing in the IMDM-dFBS(10) medium comprising0.5 mg/ml G418 and 100 nM MTX and of producing the anti-GD3 chimericantibody in a large amount were finally obtained by the same method asdescribed above. Among the obtained transformants, suitable cell lineswere selected and were made into a single cell (cloning) by limitingdilution twice. Also, using the method for determining the transcriptionproduct of an α-1,6-fucosyltransferase gene shown in Example 9, a cellline producing a relatively small amount of the transcription productwas selected and used as a suitable cell line.

(3) Preparation of Antibody-Producing Cell Using Mouse Myeloma NS0 Cell

After introducing 5 μg of the anti-GD3 chimeric antibody expressionvector pChi641LHGM4 into 4×10⁶ cells of mouse myeloma NS0 byelectroporation [Cytotechnology, 3, 133 (1990)], the cells weresuspended in 40 ml of EX-CELL302-FBS(10) (EX-CELL302 medium comprising10% FBS and 2 mM L-glutamine [hereinafter referred to as “L-Gln”;manufactured by GIBCO BRL)] and dispensed in 200 μl/well into a 96 wellculture plate (manufactured by Sumitomo Bakelite). After culturing themat 37° C. for 24 hours in a 5% CO₂ incubator, G418 was added to give aconcentration of 0.5 mg/ml, followed by culturing for 1 to 2 weeks. Theculture supernatant was recovered from wells in which colonies oftransformants showing G418 resistance were formed and growth of colonieswas observed, and the antigen binding activity of the anti-GD3 chimericantibody in the supernatant was measured by the ELISA shown in the item3 of Example 1.

Regarding the transformants in wells in which production of the anti-GD3chimeric antibody was observed in culture supernatants, in order toincrease the amount of the antibody production using a DHFR geneamplification system, each of them was suspended in anEX-CELL302-dFBS(10) medium (EX-CELL302 medium comprising 10% dFBS and 2mM L-Gln) comprising 0.5 mg/ml G418 and 50 nM MTX to give a density of 1to 2×10⁵ cells/ml, and the suspension was dispensed in 2 ml into wellsof a 24 well plate (manufactured by Greiner). Transformants showing 50nM MTX resistance were induced by culturing at 37° C. for 1 to 2 weeksin a 5% CO₂ incubator. The antigen binding activity of the anti-GD3chimeric antibody in culture supernatants in wells in which growth oftransformants was observed was measured by the ELISA shown in the item 3of Example 1. Regarding the transformants in wells in which productionof the anti-GD3 chimeric antibody was observed in culture supernatants,the MTX concentration was increased to 100 nM and then to 200 nM, andtransformants capable of growing in the EX-CELL302-dFBS(10) mediumcomprising 0.5 mg/ml G418 and 200 nM MTX and of producing the anti-GD3chimeric antibody in a large amount was finally obtained by the samemethod as described above. Among the obtained transformants, elite celllines were selected and were made into a single cell (cloning) bylimiting dilution twice. Also, using the method for determining thetranscription product of an α-1,6-fucosyltransferase gene shown inExample 9, a cell line producing a relatively small amount of thetranscription product was selected and used as a suitable cell line.

3. Measurement of Binding Activity of Antibody to GD3 (ELISA)

The binding activity of the antibody to GD3 was measured as describedbelow.

In 2 ml of ethanol solution containing 10 μg ofdipalmitoylphosphatidylcholine (manufactured by SIGMA) and 5 μg ofcholesterol (manufactured by SIGMA), 4 nmol of GD3 was dissolved. Intoeach well of a 96 well plate for ELISA (manufactured by Greiner), 20 μlof the solution (40 pmol/well in final concentration) was dispensed,followed by air-drying, 1% bovine serum albumin (hereinafter referred toas “BSA”; manufactured by SIGMA)-containing PBS (hereinafter referred toas “1% BSA-PBS”) was dispensed in 100 μl/well, and then the reaction wascarried out at room temperature for 1 hour for blocking remaining activegroups. After discarding 1% BSA-PBS, a culture supernatant of atransformant or a diluted solution of a human chimeric antibody wasdispensed in 50 μl/well to carry out the reaction at room temperaturefor 1 hour. After the reaction, each well was washed with 0.05% Tween 20(manufactured by Wako Pure Chemical Industries)-containing PBS(hereinafter referred to as “Tween-PBS”), a peroxidase-labeled goatanti-human IgG (H & L) antibody solution (manufactured by AmericanQualex) diluted 3,000 times with 1% BSA-PBS was dispensed in 50 μl/wellas a secondary antibody solution, and then the reaction was carried outat room temperature for 1 hour. After the reaction and subsequentwashing with Tween-PBS, ABTS substrate solution [solution prepared bydissolving 0.55 g of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid) ammonium salt in 1 liter of 0.1 M citrate buffer (pH 4.2) andadding 1 μl/ml of hydrogen peroxide to the solution just before use(hereinafter the same solution was used)] was dispensed in 50 μl/wellfor color development, and then absorbance at 415 nm (hereinafterreferred to as “OD415”) was measured.

4. Purification of Anti-GD3 Chimeric Antibody

(1) Culturing of Antibody-Producing Cell Derived from YB2/0 Cell andPurification of Antibody

The anti-GD3 chimeric antibody-producing transformed cell clone obtainedin the item 2(1) of Example 1 was suspended in the Hybridoma-SFM mediumcomprising 0.2% BSA, 200 nM MTX and 100 nM triiodothyronine (hereinafterreferred to as “T3”; manufactured by SIGMA) to give a density of 3×10⁵cells/ml and cultured using a 2.0 liter capacity spinner bottle(manufactured by Iwaki Glass) under agitating at a rate of 50 rpm. Afterculturing them at 37° C. for 10 days in a temperature-controlling room,the culture supernatant was recovered. The anti-GD3 chimeric antibodywas purified from the culture supernatant using a Prosep-A (manufacturedby Bioprocessing) column in accordance with the manufacture'sinstructions. The purified anti-GD3 chimeric antibody was namedYB2/0-GD3 chimeric antibody.

(2) Culturing of Antibody-Producing Cell Derived from CHO/DG44 Cell andPurification of Antibody

The anti-GD3 chimeric antibody-producing transformed cell clone obtainedin the item 2(2) of Example 1 was suspended in the EX-CELL302 mediumcomprising 3 mM L-Gln, 0.5% fatty acid concentrated solution(hereinafter referred to as “CDLC”; manufactured by GIBCO BRL) and 0.3%Pluronic F68 (hereinafter referred to as “PF68”; manufactured by GIBCOBRL) to give a density of 1×10⁶ cells/ml, and the suspension wasdispensed in 50 ml into 175 mm² flasks (manufactured by Greiner). Afterculturing them at 37° C. for 4 days in a 5% CO₂ incubator, the culturesupernatant was recovered. The anti-GD3 chimeric antibody was purifiedfrom the culture supernatant using a Prosep-A (manufactured byBioprocessing) column in accordance with the manufacture's instructions.The purified anti-GD3 chimeric antibody was named CHO/DG44-GD3 chimericantibody.

(3) Culturing of Antibody-Producing Cell Derived from NS0 Cell andPurification of Antibody

The anti-GD3 chimeric antibody-producing transformed cell clone obtainedin the item 2(3) of Example 1 was suspended in the EX-CELL302 mediumcomprising 2 mM L-Gln, 0.5 mg/ml G418, 200 nM MTX and 1% FBS, to give adensity of 1×10⁶ cells/ml, and the suspension was dispensed in 200 mlinto 175 mm² flasks (manufactured by Greiner). After culturing them at37° C. for 4 days in a 5% CO₂ incubator, the culture supernatant wasrecovered. The anti-GD3 chimeric antibody was purified from the culturesupernatant using a Prosep-A (manufactured by Bioprocessing) column inaccordance with the manufacture's instructions. The purified anti-GD3chimeric antibody was named NS0-GD3 chimeric antibody (302).

Also, the transformed cell clone was suspended in the GIT mediumcomprising 0.5 mg/ml G418 and 200 nM MTX to give a density of 3×10⁵cells/ml, and the suspension was dispensed in 200 ml into 175 mm² flasks(manufactured by Greiner). After culturing them at 37° C. for 10 days ina 5% CO₂ incubator, the culture supernatant was recovered. The anti-GD3chimeric antibody was purified from the culture supernatant using aProsep-A (manufactured by Bioprocessing) column in accordance with themanufacture's instructions. The purified anti-GD3 chimeric antibody wasnamed NS0-GD3 chimeric antibody (GIT).

(4) Culturing of Antibody-Producing Cell Derived from SP2/0 Cell andPurification of Antibody

The anti-GD3 chimeric antibody-producing transformed cell clone (KM-871(FERM BP-3512)) described in Japanese Published Unexamined PatentApplication No. 304989/93 (EP 533199) was suspended in the GIT mediumcomprising 0.5 mg/ml G418 and 200 nM MTX to give a density of 3×10⁵cells/ml, and the suspension was dispensed in 200 ml into 175 mm² flasks(manufactured by Greiner). After culturing them at 37° C. for 8 days ina 5% CO₂ incubator, the culture supernatant was recovered. The anti-GD3chimeric antibody was purified from the culture supernatant using aProsep-A (manufactured by Bioprocessing) column in accordance with themanufacture's instructions. The purified anti-GD3 chimeric antibody wasnamed SP2/0-GD3 chimeric antibody.

5. Analysis of Purified Anti-GD3 Chimeric Antibody

In accordance with a known method [Nature, 227, 680 (1970)], 4 μg ofeach of the five kinds of the anti-GD3 chimeric antibodies produced byand purified from respective animal cells, obtained in the item 4 ofExample 1, was subjected to SDS-PAGE to analyze the molecular weight andpurification degree. The results are shown in FIG. 1. As shown in FIG.1, a single band of about 150 kilodaltons (hereinafter referred to as“Kd”) in molecular weight was found under non-reducing conditions, andtwo bands of about 50 Kd and about 25 Kd under reducing conditions, ineach of the purified anti-GD3 chimeric antibodies. The molecular weightsalmost coincided with the molecular weights deduced from the cDNAnucleotide sequences of H chain and L chain of the antibody (H chain:about 49 Kd, L chain: about 23 Kd, whole molecule: about 144 Kd), andalso coincided with the reports stating that the IgG antibody has amolecular weight of about 150 Kd under non-reducing conditions and isdegraded into H chains having a molecular weight of about 50 Kd and Lchains having a molecular weight of about 25 Kd under reducingconditions due to cutting of the disulfide bond (hereinafter referred toas “S—S bond”) in the molecule [Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, Chapter 14 (1998); Monoclonal Antibodies:Principles and Practice, Academic Press Limited (1996)], so that it wasconfirmed that each anti-GD3 chimeric antibody was expressed andpurified as an antibody molecule having the true structure.

Example 2 Activity Evaluation of Anti-GD3 Chimeric Antibody 1. BindingActivity of Anti-GD3 Chimeric Antibody to GD3 (ELISA)

The activity of the five kinds of the purified anti-GD3 chimericantibodies obtained in the item 4 of Example 1 to bind to GD3(manufactured by Snow Brand Milk Products) was measured by the ELISAshown in the item 3 of Example 1. FIG. 2 shows a result of theexamination of the binding activity measured by changing theconcentration of the anti-GD3 chimeric antibody to be added. As shown inFIG. 2, the five kinds of the anti-GD3 chimeric antibodies showed almostthe same binding activity to GD3. The result shows that antigen bindingactivities of these antibodies are constant independently of theantibody-producing animal cells and their culturing methods. Also, itwas suggested from the comparison of the NS0-GD3 chimeric antibody (302)with the NS0-GD3 chimeric antibody (GIT) that the antigen bindingactivities are constant independently of the media used in theculturing.

2. In Vitro Cytotoxic Activity (ADCC Activity) of Anti-GD3 ChimericAntibody

In order to evaluate in vitro cytotoxic activity of the five kinds ofthe purified anti-GD3 chimeric antibodies obtained in the item 4 ofExample 1, the ADCC activity was measured in accordance with thefollowing method.

(1) Preparation of Target Cell Solution

A human melanoma cultured cell line G-361 (ATCC CRL 1424) was culturedusing the RPMI1640-FBS(10) medium to prepare 1×10⁶ cells, and the cellswere radioisotope-labeled by reacting them with 3.7 MBq equivalents of aradioactive substance Na₂ ⁵¹CrO₄ at 37° C. for 1 hour. After thereaction, the cells were washed three times through their suspension inthe RPMI1640-FBS(10) medium and centrifugation, re-suspended in themedium and then incubated at 4° C. for 30 minutes in ice for spontaneousdissolution of the radioactive substance. After centrifugation, theprecipitate was adjusted to 2×10⁵ cells/ml by adding 5 ml of theRPMI1640-FBS(10) medium and used as the target cell solution.

(2) Preparation of Effector Cell Solution

From a healthy person, 50 ml of venous blood was collected, and gentlymixed with 0.5 ml of heparin sodium (manufactured by TakedaPharmaceutical). The mixture was centrifuged to isolate a mononuclearcell layer using Lymphoprep (manufactured by Nycomed Pharma AS) inaccordance with the manufacture's instructions. After washing with theRPMI1640-FBS(10) medium by centrifugation three times, the resultingprecipitate was re-suspended to give a density of 2×10⁶ cells/ml usingthe medium and used as the effector cell solution.

(3) Measurement of ADCC Activity

Into each well of a 96 well U-shaped bottom plate

(manufactured by Falcon), 50 μl of the target cell solution prepared inthe above (1) (1×10⁴ cells/well) was dispensed. Next, 100 μl of theeffector cell solution prepared in the above (2) was added thereto(2×10⁵ cells/well, the ratio of effector cells to target cells becomes20:1). Subsequently, each of the anti-GD3 chimeric antibodies was addedto give a final concentration from 0.0025 to 2.5 μg/ml, followed byreaction at 37° C. for 4 hours. After the reaction, the plate wascentrifuged, and the amount of ⁵¹Cr in the supernatant was measuredusing a γ-counter. The amount of spontaneously released ⁵¹Cr wascalculated by the same operation using only the medium instead of theeffector cell solution and the antibody solution, and measuring theamount of ⁵¹Cr in the supernatant. The amount of total released ⁵¹Cr wascalculated by the same operation using only the medium instead of theantibody solution and adding 1 N hydrochloric acid instead of theeffector cell solution, and measuring the amount of ⁵¹Cr in thesupernatant. The ADCC activity was calculated from the followingequation (II):

$\begin{matrix}{{{ADCC}\mspace{14mu} {activity}\mspace{14mu} (\%)} = {\frac{{{\,^{51}{Cr}}\mspace{14mu} {in}\mspace{14mu} {sample}\mspace{14mu} {supernatant}} - {{spontaneously}\mspace{14mu} {released}\mspace{14mu} {\,^{51}{Cr}}}}{{{total}\mspace{14mu} {released}\mspace{14mu} {\,^{51}{Cr}}} - {{spontaneously}\mspace{14mu} {released}\mspace{14mu} {\,^{51}{Cr}}}} \times 100}} & ({II})\end{matrix}$

The results are shown in FIG. 3. As shown in FIG. 3, among the fivekinds of the anti-GD3 chimeric antibodies, the YB2/0-GD3 chimericantibody showed the most potent ADCC activity, followed by the SP2/0-GD3chimeric antibody, NS0-GD3 chimeric antibody and CH0-GD3 chimericantibody in that order. No difference in the ADCC activity was foundbetween the NS0-GD3 chimeric antibody (302) and NS0-GD3 chimericantibody (GIT) prepared using different media in the culturing. Theabove results show that the ADCC activity of antibodies greatly variesdepending on the kind of the animal cells to be used in theirproduction. As its mechanism, since their antigen binding activitieswere identical, it was considered that it is caused by a difference inthe structure binding to the antibody Fc region.

Example 3 Preparation of Anti-Human Interleukin 5 Receptor α Chain HumanCDR-Grafted Antibody 1. Preparation of Cell Stably Producing Anti-HumanInterleukin 5 Receptor α Chain Human CDR-Grafted Antibody (1)Preparation of Antibody-Producing Cell Using Rat Myeloma YB2/0 Cell

Using the anti-human interleukin 5 receptor a chain human CDR-graftedantibody (hereinafter referred to as “anti-hIL-5Rα CDR-graftedantibody”) expression vector, pKANTEX1259HV3LV0, described in WO97/10354, cells capable of stably producing anti-hIL-5Rα CDR-graftedantibody were prepared as described below.

After introducing 5 μg of the anti-hIL-5Rα CDR-grafted antibodyexpression vector pKANTEX1259HV3LV0 into 4×10⁶ cells of rat myelomaYB2/0 by electroporation [Cytotechnology, 3, 133 (1990)], the cells weresuspended in 40 ml of RPMI1640-FBS(10) and dispensed in 200 μl/well intoa 96 well culture plate (manufactured by Sumitomo Bakelite). Afterculturing them at 37° C. for 24 hours in a 5% CO₂ incubator, G418 wasadded to give a concentration of 0.5 mg/ml, followed by culturing for 1to 2 weeks. The culture supernatant was recovered from wells in whichcolonies of transformants showing G418 resistance were formed and growthof colonies was observed, and the antigen binding activity of theanti-hIL-5Rα CDR-grafted antibody in the supernatant was measured by theELISA shown in the item 2 of Example 3.

Regarding the transformants in wells in which production of theanti-hIL-5Rα CDR-grafted antibody was observed in culture supernatants,in order to increase amount of the antibody production using a DHFR geneamplification system, each of the them was suspended in theRPMI1640-FBS(10) medium comprising 0.5 mg/ml G418 and 50 nM MTX to givea density of 1 to 2×10⁵ cells/ml, and the suspension was dispensed in 2ml into wells of a 24 well plate (manufactured by Greiner).Transformants showing 50 nM MTX resistance were induced by culturing at37° C. for 1 to 2 weeks in a 5% CO₂ incubator. The antigen bindingactivity of the anti-hIL-5Rα CDR-grafted antibody in culturesupernatants in wells in which growth of transformants was observed wasmeasured by the ELISA shown in the item 2 of Example 3. Regarding thetransformants in wells in which production of the anti-hIL-5RαCDR-grafted antibody was observed in culture supernatants, the MTXconcentration was increased to 100 nM and then to 200 nM, andtransformants capable of growing in the RPMI1640-FBS(10) mediumcomprising 0.5 mg/ml G418 and 200 nM MTX and of producing theanti-hIL-5Rα CDR-grafted antibody in a large amount were finallyobtained in the same manner as described above. Among the obtainedtransformants, elite cell lines were selected and were made into asingle cell (cloning) by limiting dilution twice. Also, using the methodfor determining the transcription product of an α-1,6-fucosyltransferasegene shown in Example 9, a cell line producing a relatively small amountof the transcription product was selected and used as a suitable cellline. The obtained anti-hIL-5Rα CDR-grafted antibody-producingtransformed cell clone No. 3 has been deposited on Apr. 5, 1999, as FERMBP-6690 in National Institute of Bioscience and Human Technology, Agencyof Industrial Science and Technology (Higashi 1-1-3, Tsukuba, Ibaraki,Japan) (present name: International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (AIST TsukubaCentral 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken, Japan)).

(2) Preparation of Antibody-Producing Cell Using CHO/Dhfr⁻ Cell

After introducing 4 μg of the anti-hIL-5Rα CDR-grafted antibodyexpression vector pKANTEX1259HV3LV0 described in WO 97/10354 into1.6×10⁶ cells of CHO/dhfr⁻ by electroporation [Cytotechnology, 3, 133(1990)], the cells were suspended in 10 ml of IMDM-FBS(10) and dispensedin 200 μl/well into a 96 well culture plate (manufactured by IwakiGlass). After culturing them at 37° C. for 24 hours in a 5% CO₂incubator, G418 was added to give a concentration of 0.5 mg/ml, followedby culturing for 1 to 2 weeks. The culture supernatant was recoveredfrom respective well in which colonies of transformants showing G418resistance were formed and growth of colonies was observed, and theantigen binding activity of the anti-hIL-5Rα CDR-grafted antibody in thesupernatant was measured by the ELISA shown in the item 2 of Example 3.

Regarding the transformants in wells in which production of theanti-hIL-5Rα CDR-grafted antibody was observed in culture supernatants,in order to increase amount of the antibody production using a DHFR geneamplification system, each of the transformants was suspended in anIMDM-dFBS(10) medium comprising 0.5 mg/ml G418 and 10 nM MTX to give adensity of 1 to 2×10⁵ cells/ml, and the suspension was dispensed in 0.5ml into wells of a 24 well plate (manufactured by Iwaki Glass).Transformants showing 10 nM MTX resistance were induced by culturing at37° C. for 1 to 2 weeks in a 5% CO₂ incubator. Regarding thetransformants in wells in which their growth was observed, the MTXconcentration was increased to 100 nM and then to 500 nM, andtransformants capable of growing in the IMDM-dFBS(10) medium comprising0.5 mg/ml G418 and 500 nM MTX and of producing the anti-hIL-5RαCDR-grafted antibody in a large amount were finally obtained in the samemanner as described above. Among the obtained transformants, elite celllines were selected and were made into a single cell (cloning) bylimiting dilution twice. Also, using the method for determining thetranscription product of an α-1,6-fucosyltransferase gene shown inExample 9, a cell line producing a relatively small amount of thetranscription product was selected and used as a suitable cell line.

(3) Production of Antibody-Producing Cell Using Mouse myeloma NS0 cell

An anti-hIL-5Rα CDR-grafted antibody expression vector was prepared inaccordance with the method of Yarranton et al. [BIO/TECHNOLOGY, 10, 169(1992)] and using the antibody H chain cDNA and L chain cDNA on theanti-hIL-5Rα CDR-grafted antibody expression vector pKANTEX1259HV3LV0described in WO 97/10354, and NS0 cell was transformed to obtaintransformants capable of producing the anti-hIL-5Rα CDR-grafted antibodyin a large amount. Among the obtained transformants, elite cell lineswere selected and were made into a single cell (cloning) by limitingdilution twice. Also, using the method for determining the transcriptionproduction of an α-1,6-fucosyltransferase gene shown in Example 9, acell line producing a relatively small amount of the transcriptionproduct was selected and used as a suitable cell line.

2. Measurement of Binding Activity of Antibody to hIL-5Rα (ELISA)

The binding activity of the antibody to hIL-5Rα was measured asdescribed below.

A solution was prepared by diluting the anti-hIL-5Rα mouse antibodyKM1257 described in WO 97/10354 with PBS to give a concentration of 10μg/ml, and 50 μl of the resulting solution was dispensed into each wellof a 96 well plate for ELISA (manufactured by Greiner), followed byreaction at 4° C. for 20 hours. After the reaction, 1% BSA-PBS wasdispensed in 100 μl/well, and then the reaction was carried out at roomtemperature for 1 hour to block remaining active groups. Afterdiscarding 1% BSA-PBS, a solution prepared by diluting the solublehIL-5Rα described in WO 97/10354 with 1% BSA-PBS to give a concentrationof 0.5 μg/ml was dispensed in 50 μl/well, followed by reaction at 4° C.for 20 hours. After the reaction, each well was washed with Tween-PBS,culture supernatants of transformants or diluted solutions of a purifiedhuman CDR-grafted antibodies were dispensed in 50 μg/well to carry outthe reaction at room temperature for 2 hours. After the reaction, eachwell was washed with Tween-PBS, a peroxidase-labeled goat anti-human IgG(H & L) antibody solution (manufactured by American Qualex) diluted3,000 times with 1% BSA-PBS was dispensed in 50 μl/well as a secondaryantibody solution, followed by reaction at room temperature for 1 hour.After the reaction and subsequent washing with Tween-PBS, the ABTSsubstrate solution was dispensed in 50 μl/well for color development,and then the absorbance at OD415 was measured.

3. Purification of Anti-hIL-5Rα CDR-Grafted Antibody

(1) Culturing of Antibody-Producing Cell Derived from YB2/0 Cell andPurification of Antibody

The anti-hIL-5Rα CDR-grafted antibody-producing transformed cell cloneobtained in the item 1(1) of Example 3 was suspended in the GIT mediumcomprising 0.5 mg/ml G418 and 200 nM MTX to give a density of 3×10⁵cells/ml and dispensed in 200 ml into 175 mm² flasks (manufactured byGreiner). After culturing them at 37° C. for 8 days in a 5% CO₂incubator, the culture supernatant was recovered. The anti-hIL-5RαCDR-grafted antibody was purified from the culture supernatant using ionexchange chromatography and a gel filtration method. The purifiedanti-hIL-5Rα CDR-grafted antibody was named YB2/0-hIL-5R CDR antibody.

(2) Culturing of Antibody-Producing Cell Derived from CHO/dhfr⁻ Cell andPurification of Antibody

The anti-hIL-5Rα CDR-grafted antibody-producing transformed cell cloneobtained in the item 1(2) of Example 3 was suspended in the EX-CELL302medium comprising 3 mM L-Gln, 0.5% CDLC and 0.3% PF68 to give a densityof 3×10⁵ cells/ml and cultured using a 4.0 liter capacity spinner bottle(manufactured by Iwaki Glass) under agitating at a rate of 100 rpm.After culturing them at 37° C. for 10 days in a temperature-controllingroom, the culture supernatant was recovered. The anti-hIL-5RαCDR-grafted antibody was purified from the culture supernatant using ionexchange chromatography and a gel filtration method. The purifiedanti-hIL-5Rα CDR-grafted antibody was named CHO/d-hIL-5R CDR antibody.

(3) Culturing of Antibody-Producing Cell Derived from NS0 Cell andPurification of Antibody

The anti-hIL-5Rα CDR-grafted antibody-producing transformed cell cloneobtained in the item 1(3) of Example 3 was cultured in accordance withthe method of Yarranton et al. [BIO/TECHNOLOGY, 10, 169 (1992)] and thena culture supernatant was recovered. The anti-hIL-5Rα CDR-graftedantibody was purified from the culture supernatant using ion exchangechromatography and the gel filtration method. The purified anti-hIL-5RαCDR-grafted antibody was named NS0-hIL-5R CDR antibody.

4. Analysis of Purified Anti-hIL-5Rα CDR-Grafted Antibodies

In accordance with a known method [Nature, 227, 680 (1970)], 4 μg ofeach of the three kinds of the anti-hIL-5Rα CDR-grafted antibodiesproduced by and purified from each animal cells, obtained in the item 3of Example 3, was subjected to SDS-PAGE to analyze the molecular weightand purification degree. The results are shown in FIG. 4. As shown inFIG. 4, a single band of about 150 Kd in molecular weight was foundunder non-reducing conditions, and two bands of about 50 Kd and about 25Kd under reducing conditions, in each of the purified anti-hIL-5RαCDR-grafted antibodies. The molecular weights almost coincided with themolecular weights deduced from the cDNA nucleotide sequences of H chainand L chain of the antibody (H chain: about 49 Kd, L chain: about 23 Kd,whole molecule: about 144 Kd), and also coincided with the reportsstating that the IgG antibody has a molecular weight of about 150 Kdunder non-reducing conditions and is degraded into H chains having amolecular weight of about 50 Kd and L chains having a molecular weightof about 25 Kd under reducing conditions due to cutting of the S—S bondin the molecule [Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, Chapter 14 (1998); Monoclonal Antibodies: Principles andPractice, Academic Press Limited (1996)], so that it was confirmed thateach anti-hIL-5Rα CDR-grafted antibody was expressed and purified as anantibody molecule having the true structure.

Example 4 Activity Evaluation of Anti-hIL-5Rα CDR-Grafted Antibody

1. Binding Activity of Anti-hIL-5Rα CDR-Grafted Antibody to hIL-5Rα(ELISA)

The activity of the three kinds of the purified anti-hIL-5Rα CDR-graftedantibodies obtained in the item 3 of Example 3 to bind to hIL-5Rα wasmeasured by the ELISA shown in the item 2 of Example 3. FIG. 5 shows aresult of the examination of the binding activity measured by changingconcentration of the anti-hIL-5Rα CDR-grafted antibody to be added. Asshown in FIG. 5, the three kinds of the anti-hIL-5Rα CDR-graftedantibodies showed almost the same binding activity to hIL-5Rα. Theresult shows that the antigen binding activities of these antibodies areconstant independently of the antibody-producing animal cells and theirculturing methods, similar to the result of the item 1 of Example 2.

2. In Vitro Cytotoxic Activity (ADCC Activity) of Anti-hIL-5RαCDR-Grafted Antibody

In order to evaluate in vitro cytotoxic activity of the three kinds ofthe purified anti-hIL-5Rα CDR-grafted antibodies obtained in the item 3of Example 3, the ADCC activity was measured in accordance with thefollowing method.

(1) Preparation of Target Cell Solution

A mouse T cell line CTLL-2(h5R) expressing the hIL-5Rα chain and β chaindescribed in WO 97/10354 was cultured using the RPMI1640-FBS(10) mediumto give a density of 1×10⁶ cells/0.5 ml, and the cells wereradioisotope-labeled by reacting them with 3.7 MBq equivalents of aradioactive substance Na₂ ⁵¹CrO₄ at 37° C. for 1.5 hours. After thereaction, the cells were washed three times through their suspension inthe RPMI1640-FBS(10) medium and centrifugation, resuspended in themedium and then incubated at 4° C. for 30 minutes in ice for spontaneousdissolution of the radioactive substance. After the centrifugation, theprecipitate was adjusted to give a density of 2×10⁵ cells/ml by adding 5ml of the RPMI1640-FBS(10) medium and used as the target cell solution.

(2) Preparation of Effector Cell Solution

From a healthy person, 50 ml of venous blood was collected and gentlymixed with 0.5 ml of heparin sodium (manufactured by TakedaPharmaceutical). The mixture was centrifuged to separate a mononuclearcell layer using Polymorphprep (manufactured by Nycomed Pharma AS) andin accordance with the manufacture's instructions. After washing withthe RPMI1640-FBS(10) medium by centrifugation three times, the resultingcells were resuspended to give a density of 9×10⁶ cells/ml using themedium and used as the effector cell solution.

(3) Measurement of ADCC Activity

Into each well of a 96 well U-shaped bottom plate (manufactured byFalcon), 50 μl of the target cell solution prepared in the above (1)(1×10⁴ cells/well) was dispensed. Next, 100 μl of the effector cellsolution prepared in the above (2) was dispensed (9×10⁵ cells/well, theratio of effector cells to target cells becomes 90:1). Subsequently,each of the anti-hIL-5Rα CDR-grafted antibodies was added to give afinal concentration of 0.001 to 0.1 μg/ml, followed by reaction at 37°C. for 4 hours. After the reaction, the plate was centrifuged, and theamount of ⁵¹Cr in the supernatant was measured using a γ-counter. Theamount of spontaneously released ⁵¹Cr was calculated by the sameoperation using only the medium instead of the effector cell solutionand the antibody solution, and measuring the amount of ⁵¹Cr in thesupernatant. The amount of total released ⁵¹Cr was calculated by thesame operation using only the medium instead of the antibody solutionand adding 1 N hydrochloric acid instead of the effector cell solution,and measuring the amount of ⁵¹Cr in the supernatant.

The ADCC activity was calculated from the above equation (II).

The results are shown in FIG. 6. As shown in FIG. 6, among the threekinds of the anti-hIL-5Rα CDR-grafted antibodies, the YB2/0-hIL-5R CDRantibody showed the most potent ADCC activity, followed by theCHO/d-hIL-5R CDR antibody and the NS0-hIL-5R CDR antibody in this order.Similar to the result of the item 2 of Example 2, the above results showthat the ADCC activity of antibodies greatly varies depending on theanimal cells to be used in their production. In addition, since theantibodies produced by the YB2/0 cell showed the most potent ADCCactivity in both cases of the two kinds of the humanized antibodies, itwas revealed that an antibody having potent ADCC activity can beproduced by using the YB2/0 cell.

3. In Vivo Activity Evaluation of Anti-hIL-5Rα CDR-Grafted Antibody

In order to evaluate in vivo activity of the three kinds of the purifiedanti-hIL-5Rα CDR-grafted antibodies obtained in the item 3 of Example 3,the inhibition activity in an hIL-5-induced eosinophilia increasingmodel of Macaca faseicularis was examined in accordance with thefollowing method.

The hIL-5 (preparation method is described in WO 97/10354) wasadministered to Macaca faseicularis under the dorsal skin at a dose of 1μg/kg, starting on the first day and once a day for a total of 14 times.Each anti-hIL-5Rα CDR-grafted antibody was intravenously administered ata dose of 0.3 mg/kg one hour before the hIL-5 administration on the dayzero. An antibody-non-added group was used as the control. In theantibody-administered groups, three animals of Macaca faseicularis wereused in each group (No. 301, No. 302, No. 303, No. 401, No. 402, No.403, No. 501, No. 502 and No. 503), and two animals (No. 101 and No.102) were used in the antibody-non-added group. Starting 7 days beforecommencement of the administration and until 42 days after theadministration, about 1 ml of blood was periodically collected from asaphena or a femoral vein, and the number of eosinophils in 1 μl ofperipheral blood was measured. The results are shown in FIG. 7. As shownin FIG. 7, increase in the blood eosinophil was completely inhibited inthe group to which the YB2/0-hIL-5R CDR antibody was administered. Onthe other hand, complete inhibition activity was found in one animal inthe group to which the CHO/d-hIL-5R CDR antibody was administered, butthe inhibition activity was not sufficient in two animals. In the groupto which NS0-hIL-5R CDR antibody was administered, complete inhibitionactivity was not found and its effect was not sufficient.

The above results show that the in vivo activity of antibodies greatlyvaries depending on the animal cells to be used in their production. Inaddition, since a positive correlation was found between the degree ofthe in vivo activity of the anti-hIL-5Rα CDR-grafted antibody and thedegree of its ADCC activity described in the item 2 of Example 4, it wasindicated that the degree of ADCC activity is remarkably important forits activity expression.

Based on the above results, it is expected that an antibody havingpotent ADCC activity is useful also in the clinical field for variousdiseases in human.

Example 5 Analysis of Sugar Chain which Enhances ADCC Activity 1.Preparation of 2-Aminopyridine-Labeled Sugar Chain (PA-Treated SugarChain)

The humanized antibody of the present invention was acid-hydrolyzed withhydrochloric acid to remove sialic acid. After hydrochloric acid wascompletely removed, the sugar chain was cleaved from the protein byhydrazinolysis [Method of Enzymology, 83, 263 (1982)]. Hydrazine wasremoved, and N-acetylation was carried out by adding an aqueous ammoniumacetate solution and acetic anhydride. After lyophilizing, fluorescencelabeling with 2-aminopyridine was carried out [J. Biochem., 95, 197(1984)]. The fluorescence-labeled sugar chain (PA-treated sugar chain)was separated from impurity using Surperdex Peptide HR 10/30 Column(manufactured by Pharmacia). The sugar chain fraction was dried using acentrifugal concentrator and used as a purified PA-treated sugar chain.

2. Reverse Phase HPLC Analysis of PA-Treated Sugar Chain of PurifiedAnti-hIL-5Rα CDR-Grafted Antibody

According to the method in the item 1 of Example 5, various anti-hIL-5RαCDR-grafted antibodies produced in Example 3 were subjected toPA-treated sugar chain treatment, and reverse phase HPLC analysis wascarried out by CLC-ODS column (manufactured by Shimadzu). An excessamount of α-L-fucosidase (derived from bovine kidney, manufactured bySIGMA) was added to the PA-treated sugar chain for digestion (37° C., 15hours), and then the products were analyzed by reverse phase HPLC (FIG.8). It was confirmed that the asparagine-linked sugar chain is elutedfor 30 minutes to 80 minute using PA-treated sugar chain standardsmanufactured by Takara Shuzo. The ratio of sugar chains whose reversephase HPLC elution positions were shifted (sugar chains eluted for 48minutes to 78 minutes) by the α-L-fucosidase digestion was calculated.The results are shown in Table 1.

TABLE 1 Antibody-producing cell α-1,6-Fucose-linked sugar chain (%)YB2/0 47 NS0 73

About 47% of the anti-hIL-5R CDR-grafted antibody produced by the YB2/0cell and about 73% of the anti-hIL-5R CDR-grafted antibody produced bythe NS0 cell were sugar chains in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe N-glycoside-linked sugar chain (hereinafter referred to as “sugarchain having α-1,6-fucose”). Thus, the ratio of sugar chains in which1-position of fucose is not bound to 6-position of N-acetylglucosaminein the reducing end through α-bond in the N-glycoside-linked sugar chain(hereinafter referred to as “α-1,6-fucose-free sugar chain”) is higherin the antibody produced by the YB2/0 cell than in the antibody producedby the NS0 cell.

3. Analysis of Monosaccharide Composition of Purified Anti-hIL-5RαCDR-Grafted Antibody

Sugar chains of anti-hIL-5Rα CDR-grafted antibodies produced by theYB2/0 cell, NS0 cell and CHO/d cell were hydrolyzed into monosaccharidesby acid hydrolysis with trifluoroacetic acid, and monosaccharidecomposition analysis was carried out using BioLC (manufactured byDionex).

Among N-glycoside-linked sugar chains, there are 3 mannose units in onesugar chain in the complex type N-glycoside-linked sugar chain. Arelative ratio of each monosaccharide obtained by calculating the numberof mannose as 3 is shown in Table 2.

TABLE 2 Antibody-producing ADCC activity cell Fuc GlcNAc Gal Man (%)*YB2/0 0.60 4.98 0.30 3.00 42.27 NS0 1.06 3.94 0.66 3.00 16.22 CHO/dhFr⁻0.85 3.59 0.49 3.00 25.73 CHO/dhFr⁻ 0.91 3.80 0.27 3.00 25.73 *Antibodyconcentration: 0.01 μg/ml

Since the relative ratios of fucose were in an order of YB2/0<CHO/d<NS0,the sugar chain produced in the antibody produced by YB2/0 cell showedthe lowest fucose content as also shown in the present results.

4. Sugar Chain Analysis of Antibody Produced by CHO/Dhfr⁻ Cell

PA-treated sugar chains were prepared from purified anti-hIL-5RαCDR-grafted antibody produced by CHO/dhfr⁻ cell, and reverse phase HPLCanalysis was carried out using CLC-ODS column (manufactured by Shimadzu)(FIG. 9). In FIG. 9, an elution time from 35 to 45 minutes correspondedto sugar chains having no fucose and an elution time from to 60 minutescorresponded to sugar chains having fucose. Similar to the case of theantibody produced by mouse myeloma NS0 cell, the anti-hIL-5RαCDR-grafted antibody produced by CHO/dhfr⁻ cell had less fucose-freesugar chain content than the antibody produced by rat myeloma YB2/0cell.

Example 6 Separation of Potent ADCC Activity Antibody

The anti-hIL-5Rα CDR-grafted antibody produced by rat myeloma YB2/0 cellwas separated using a lectin column which binds to sugar chains havingfucose. HPLC was carried out using LC-6A manufactured by Shimadzu at aflow rate of 1 ml/min and at room temperature as the column temperature.After equilibration with 50 mM Tris-sulfate buffer (pH 7.3), thepurified anti-hIL-5Rα CDR-grafted antibody was injected and then elutedby a linear density gradient (60 minutes) of 0.2 M α-methylmannoside(manufactured by Nakalai Tesque). The anti-hIL-5Rα CDR-grafted antibodywas separated into non-adsorbed fraction and adsorbed fraction. When thenon-adsorbed fraction and a part of the adsorbed fraction were sampledand their binding activity to hIL-5Rα was measured, they showed similarbinding activity (FIG. 10A). When the ADCC activity was measured, thenon-adsorbed fraction showed potent ADCC activity (100 to 1000 folds)than that of the part of adsorbed fraction (FIG. 10B). In addition,PA-treated sugar chains were prepared from the non-adsorbed fraction anda part of the adsorbed fraction, and reverse HPLC analysis was carriedout using CLC-ODS column (manufactured by Shimadzu) (FIG. 11). In thenon-adsorbed fraction, an antibody binding to fucose-free sugar chainswas mainly present, and in the part of adsorbed fraction, an antibodybinding to sugar chains having fucose was mainly present.

Example 7 Activity Evaluation of Anti-GD3 Chimeric Antibody HavingDifferent Ratio of α-1,6-Fucose-Free Sugar Chain 1. Preparation ofAnti-GD3 Chimeric Antibodies Having Different Ratio of α-1,6-Fucose-FreeSugar Chain

In accordance with the method described in the item 2(1) of Example 1,transformed clones derived from YB2/0 cell capable of producing ananti-GD3 chimeric antibody was obtained. Antibodies were prepared fromthe transformed clones derived from YB2/0 cell and named lot 1, lot 2and lot 3. Each sugar chain which is bound to the anti-GD3 chimericantibodies of lot 1, lot 2 and lot 3 was analyzed in accordance with themethod of Example 11(6), and it was found that the ratios ofα-1,6-fucose-free sugar chains were 50%, 45% and 29%, respectively.Herein, these samples are referred to as anti-GD3 chimeric antibody(50%), anti-GD3 chimeric antibody (45%) and anti-GD3 chimeric antibody(29%).

Also, sugar chains of the anti-GD3 chimeric antibody derived from theCHO/DG44 cell prepared in the item 2(2) of Example 1 were analyzed inaccordance with the method of Example 11(6), and it was found that theratio of α-1,6-fucose-free sugar chains was 7%. Herein, the sample isreferred to as anti-GD3 chimeric antibody (7%).

The anti-GD3 chimeric antibody (45%) and anti-GD3 chimeric antibody (7%)were mixed at a ratio of anti-GD3 chimeric antibody (45%): anti-GD3chimeric antibody (7%)=5:3 or 1:7. Sugar chains of the samples wereanalyzed in accordance with the method of Example 10(6), and it wasfound that samples having the ratio of α-1,6-fucose-free sugar chains of24% and 13% (calculated value) were prepared. Herein, they are referredto as anti-GD3 chimeric antibody (24%) and anti-GD3 chimeric antibody(13%).

Results of the sugar chain analysis of each of the samples are shown inFIG. 12. The ratio of α-1,6-fucose-free sugar chains was shown as anaverage value of the result of two sugar chain analyses.

2. Evaluation of Binding Activity to GD3 (ELISA)

The binding activities of the six kinds of the anti-GD3 chimericantibodies having a different ratio of α-1,6-fucose-free sugar chainsprepared in the item 1 of Example 7 against GD3 (manufactured by SnowBrand Milk Products) were measured by the ELISA shown in the item 3 ofExample 1. As a result, all of the six kinds of the anti-GD3 chimericantibodies showed almost the same GD3-binding activity as shown in FIG.13, and it was found that the ratio of the α-1,6-fucose-free sugarchains does not have influence on the antigen binding activity of theantibody.

3. Evaluation of ADCC Activity on Human Melanoma Cell Line

The ADCC activity of anti-GD3 chimeric antibodies on a human melanomacell line G-361 (ATCC CRL 1424) was measured as follows.

(1) Preparation of Target Cell Suspension

1×10⁶ cells of a human melanoma cell line G-361 were prepared, a 3.7 MBqequivalent of a radioactive substance Na₂ ⁵¹CrO₄ was added thereto andthe mixture was allowed to react at 37° C. for 1 hour to label the cellswith the radioisotope. After the reaction, the cells were washed threetimes by a procedure of their suspension in a medium and subsequentcentrifugation, re-suspended in the medium and then incubated at 4° C.for 30 minutes in ice to effect spontaneous dissociation of theradioactive substance. After centrifugation, the cells were adjusted to2×10⁵ cells/ml by adding 5 ml of the medium and used as a target cellsuspension.

(2) Preparation of Human Effector Cell Suspension

A 50 ml portion of peripheral blood was collected from a healthy personand gently mixed with 0.5 ml of heparin sodium (manufactured by ShimizuPharmaceutical). Using Lymphoprep (manufactured by AXIS SHIELD), thiswas centrifuged (800 g, 20 minutes) in accordance with the manufacture'sinstructions to separate a mononuclear cell layer. The cells were washedby centrifuging (1,200 rpm, 5 minutes) three times using a medium andthen re-suspended in the medium to give a density of 2×10⁶ cells/ml andused as a human effector cell suspension.

(3) Measurement of ADCC Activity

The target cell suspension prepared in the (1) was dispensed in 50 μl(1×10⁴ cells/well) into each well of a 96 well U-bottom plate(manufactured by Falcon). Next, 100 μl of the human effector cellsuspension prepared in the (2) was added thereto (2×10⁵ cells/well,ratio of the human effector cells to the target cells was 20:1). Each ofthe anti-GD3 chimeric antibodies was added thereto to give a finalconcentration of 0.0005 to 5 μg/ml, followed by reaction at 37° C. for 4hours. After the reaction, the plate was centrifuged and the amount of⁵¹Cr in the supernatant was measured using a γ-counter. An amount of thespontaneously dissociated ⁵¹Cr was calculated by carrying out the sameprocedure using the medium alone instead of the human effector cellsuspension and antibody solution, and measuring the amount of ⁵¹Cr inthe supernatant. An amount of the total dissociated ⁵¹Cr was calculatedby carrying out the same procedure using the medium alone instead of theantibody solution and a 1 mol/l hydrochloric acid solution instead ofthe human effector cell suspension, and measuring the amount of ⁵¹Cr inthe supernatant. The cytotoxic activity (%) was calculated usingequation (II).

FIGS. 14 and 15 show results of the measurement of ADCC activity of thesix kinds of the anti-GD3 chimeric antibodies having a different ratioof α-1,6-fucose-free sugar chains at various concentrations (0.0005 to 5μg/ml) using effector cells of two healthy donors (A and B),respectively. As shown in FIGS. 14 and 15, ADCC activity of the anti-GD3chimeric antibodies showed a tendency to increase in proportion to theratio of α-1,6-fucose-free sugar chains at each antibody concentration.The ADCC activity decreases when the antibody concentration is low. Atan antibody concentration of 0.05 μg/ml, the antibody in which the ratioof α-1,6-fucose-free sugar chains is 24%, 29%, 45% or 50% showed almostthe same potent ADCC activity but the ADCC activity was low in theantibody (13%) or (7%) in which the ratio of α-1,6-fucose-free sugarchains is less that 20%. These results were the same when the effectorcell donor was changed.

Example 8 Activity Evaluation of Anti-CCR4 Chimeric Antibody HavingDifferent Ratio of α-1,6-Fucose-Free Sugar Chain 1. Production of CellsStably Producing Anti-CCR4 Chimeric Antibody

Cells which capable of stably producing an anti-CCR4 chimeric antibodywere prepared as follows using a tandem type expression vectorpKANTEX2160 for an anti-CCR4 chimeric antibody described in WO 01/64754.

(1) Preparation of Antibody-Producing Cell Using Rat Myeloma YB2/0 Cell

After introducing 10 μg of the anti-CCR4 chimeric antibody expressionvector pKANTEX2160 into 4×10⁶ cells of rat myeloma YB2/0 cell (ATCC CRL1662) by electroporation [Cytotechnology, 3, 133 (1990)], the cells weresuspended in 40 ml of Hybridoma-SFM-FBS(5) [Hybridoma-SFM medium(manufactured by Invitrogen) comprising 5% FBS (manufactured by PAALaboratories)] and dispensed in 200 μl/well into a 96 well culture plate(manufactured by Sumitomo Bakelite). After culturing them at 37° C. for24 hours in a 5% CO₂ incubator, G418 was added to give a concentrationof 1 mg/ml, followed by culturing for 1 to 2 weeks. Culture supernatantwas recovered from wells in which growth of transformants showing G418resistance was observed by the formation of colonies, and antigenbinding activity of the anti-CCR4 chimeric antibody in the supernatantwas measured by the ELISA described in the item 2 of Example 8.

Regarding the transformants in wells in which production of theanti-CCR4 chimeric antibody was observed in culture supernatants, inorder to increase an amount of the antibody production using a DHFR geneamplification system, each of them was suspended in theHybridoma-SFM-FBS(5) medium comprising 1 mg/ml G418 and 50 nM DHFRinhibitor MTX (manufactured by SIGMA) to give a density of 1 to 2×10⁵cells/ml, and the suspension was dispensed in 1 ml portions into wellsof a 24 well plate (manufactured by Greiner). After culturing them at37° C. for 1 to 2 weeks in a 5% CO₂ incubator, transformants showing 50nM MTX resistance were induced. Antigen binding activity of theanti-CCR4 chimeric antibody in culture supernatants in wells in whichgrowth of transformants was observed was measured by the ELISA describedin the item 2 of Example 8.

Regarding the transformants in wells in which production of theanti-CCR4 chimeric antibody was observed in culture supernatants, theMTX concentration was increased by the same method, and a transformantcapable of growing in the Hybridoma-SFM-FBS(5) medium comprising 200 nMMTX and of producing the anti-CCR4 chimeric antibody in a large amountwas finally obtained. The obtained transformant was made into a singlecell (cloning) by limiting dilution twice, and the obtained cloned cellline was named KM2760 #58-35-16. In this case, using the method fordetermining the transcription product of an α-1,6-fucosyltransferasegene shown in Example 9, a cell line producing a relatively small amountof the transcription product was selected and used as a suitable cellline.

(2) Preparation of Antibody-Producing Cell Using CHO/DG44 Cell

After introducing 4 μg of the anti-CCR4 chimeric antibody expressionvector pKANTEX2160 into 1.6×10⁶ cells of CHO/DG44 cell byelectroporation [Cytotechnology, 3, 133 (1990)], the cells weresuspended in 10 ml of IMDM-dFBS(10)-HT(1) [IMDM medium (manufactured byInvitrogen) comprising 10% dFBS (manufactured by Invitrogen) and 1×concentration of HT supplement (manufactured by Invitrogen)] anddispensed in 100 μl/well into a 96 well culture plate (manufactured byIwaki Glass). After culturing them at 37° C. for 24 hours in a 5% CO₂incubator, the medium was changed to IMDM-dFBS(10) (IMDM mediumcomprising 10% of dialyzed FBS), followed by culturing for 1 to 2 weeks.Culture supernatant was recovered from wells in which the growth wasobserved due to formation of a transformant showing HT-independentgrowth, and an expression level of the anti-CCR4 chimeric antibody inthe supernatant was measured by the ELISA described in the item 2 ofExample 8.

Regarding the transformants in wells in which production of theanti-CCR4 chimeric antibody was observed in culture supernatants, inorder to increase an amount of the antibody production using a DHFR geneamplification system, each of them was suspended in the IMDM-dFBS(10)medium comprising 50 nM MTX to give a density of 1 to 2×10⁵ cells/ml,and the suspension was dispensed in 0.5 ml into wells of a 24 well plate(manufactured by Iwaki Glass). After culturing them at 37° C. for 1 to 2weeks in a 5% CO₂ incubator, transformants showing 50 nM MTX resistancewere induced. Regarding the transformants in wells in which the growthwas observed, the MTX concentration was increased to 200 nM by the samemethod, and a transformant capable of growing in the IMDM-dFBS(10)medium comprising 200 nM MTX and of producing the anti-CCR4 chimericantibody in a large amount was finally obtained. The obtainedtransformant was named 5-03. In this case, using the method fordetermining the transcription product of an α-1,6-fucosyltransferasegene shown in Example 9, a cell line producing a relatively small amountof the transcription product was selected and used as a suitable cellline.

2. Antibody Binding Activity to CCR4 Partial Peptide (ELISA)

Compound 1 (SEQ ID NO:25) was selected as a human CCR4 extracellularregion peptide capable of reacting with the anti-CCR4 chimeric antibody.In order to use it in the activity measurement by ELISA, a conjugatewith BSA (bovine serum albumin) (manufactured by Nakalai Tesque) wasprepared by the following method and used as the antigen. That is, 100ml of a DMSO solution comprising 25 mg/ml SMCC[4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acidN-hydroxysuccinimide ester] (manufactured by Sigma) was added dropwiseto 900 ml of a 10 mg BSA-containing PBS solution under stirring using avortex, followed by gently stirring for 30 minutes. A 1 ml portion ofthe reaction solution was applied to a gel filtration column such asNAP-10 column or the like equilibrated with 25 ml of PBS, and theneluted with 1.5 ml of PBS and the resulting eluate was used as aBSA-SMCC solution (BSA concentration was calculated based on A₂₈₀measurement). Next, 250 ml of PBS was added to 0.5 mg of Compound 1 andthen completely dissolved by adding 250 ml of DMF, and the BSA-SMCCsolution was added thereto under vortex, followed by gently stirring for3 hours. The reaction solution was dialyzed against PBS at 4° C.overnight, sodium azide was added thereto to give a final concentrationof 0.05%, and the mixture was filtered through a 0.22 mm filter to beused as a BSA-compound 1 solution.

The prepared conjugate was dispensed at 0.05 μg/ml and 50 μl/well into a96 well EIA plate (manufactured by Greiner) and incubated for adhesionat 4° C. overnight. After washing each well with PBS, 1% BSA-PBS wasadded thereto in 100 μl/well and allowed to react at room temperature toblock the remaining active groups. After washing each well with PBScontaining 0.05% Tween 20 (hereinafter referred to as “Tween-PBS”), aculture supernatant of a transformant was added at 50 μl/well andallowed to react at room temperature for 1 hour. After the reaction,each well was washed with Tween-PBS, and then a peroxidase-labeled goatanti-human IgG(γ) antibody solution (manufactured by American Qualex)diluted 6000 times with 1% BSA-PBS as the secondary antibody was addedat 50 μl/well and allowed to react at room temperature for 1 hour. Afterthe reaction and subsequent washing with Tween-PBS, the ABTS substratesolution was added at 50 μl/well for color development, and 20 minutesthereafter, the reaction was stopped by adding a 5% SDS solution at 50μl/well. Thereafter, the absorbance at OD415 was measured. The anti-CCR4chimeric antibody obtained in the item 1 of Example 8 showed the bindingactivity to CCR4.

3. Purification of Anti-CCR4 Chimeric Antibody

(1) Culturing of Antibody-Producing Cell Derived from YB2/0 Cell andPurification of Antibody

The anti-CCR4 chimeric antibody-expressing transformant cell cloneKM2760 #58-35-16 obtained in the item 1(1) of Example 8 was suspended inHybridoma-SFM (manufactured by Invitrogen) medium comprising 200 nM MTXand 5% of Daigo's GF21 (manufactured by Wako Pure Chemical Industries)to give a density of 2×10⁵ cells/ml and subjected to fed-batch shakingculturing using a spinner bottle (manufactured by Iwaki Glass) in aconstant temperature chamber of 37° C. After culturing them for 8 to 10days, the anti-CCR4 chimeric antibody was purified from the culturesupernatant recovered using Prosep-A (manufactured by Millipore) columnand gel filtration. The purified anti-CCR4 chimeric antibody was namedKM2760-1.

(2) Culturing of Antibody-Producing Cell Derived from CHO-DG44 Cell andPurification of Antibody

The anti-CCR4 chimeric antibody-producing transformant cell line 5-03obtained in the item 1(2) of Example 8 was cultured at 37° C. in a 5%CO₂ incubator using IMDM-dFBS(10) medium in a 182 cm² flask(manufactured by Greiner). When the cell density reached confluent afterseveral days, the culture supernatant was discarded, and the cells werewashed with 25 ml of PBS buffer and then mixed with 35 ml of EXCELL 301medium (manufactured by JRH). After culturing them at 37° C. for 7 daysin a 5% CO₂ incubator, the culture supernatant was recovered. Theanti-CCR4 chimeric antibody was purified from the culture supernatantusing Prosep-A (manufactured by Millipore) column in accordance with themanufacture's instructions. The purified anti-CCR4 chimeric antibody wasnamed KM3060.

When the binding activity to CCR4 of KM2760-1 and KM3060 was measured byELISA, they showed equivalent binding activity.

4. Analysis of Purified Anti-CCR4 Chimeric Antibodies

Each (4 μg) of the two kinds of the anti-CCR4 chimeric antibodiesproduced by and purified from in different animal cells, obtained in theitem 1 of this Example was subjected to SDS-PAGE in accordance with aknown method [Nature, 227, 680 (1970)], and the molecular weight andpurification degree were analyzed. In each of the purified anti-CCR4chimeric antibodies, a single band corresponding to the molecular weightof about 150 Kd was found under non-reducing conditions, and two bandsof about 50 Kd and about 25 Kd were found under reducing conditions. Themolecular weights almost coincided with the molecular weights deducedfrom the cDNA nucleotide sequences of antibody H chain and L chain (Hchain: about 49 Kd, L chain: about 23 Kd, whole molecule: about 144 Kd)and coincided with reports stating that an IgG type antibody has amolecular weight of about 150 Kd under non-reducing conditions and isdegraded into H chain having a molecular weight of about 50 Kd and Lchain having a molecular weight of about 25 Kd under reducing conditionscaused by cutting an S—S bond in the molecule [Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, Chapter 14 (1988), MonoclonalAntibodies: Principles and Practice, Academic Press Limited (1996)],thus confirming that the anti-CCR4 chimeric antibody was expressed andpurified as an antibody molecule having a correct structure.

5. Preparation of Anti-CCR4 Chimeric Antibody Having Different Ratio ofα-1,6-Fucose-Free Sugar Chain

Sugar chains which are bound to anti-CCR4 chimeric antibody KM2760-1derived from YB2/0 cell prepared in the item 3(1) of Example 8 and theanti-CCR4 chimeric antibody KM3060 derived from CHO/DG44 cell preparedin the item 3(2) of Example 8 were analyzed in accordance with themethod of Example 10(6). The ratio of α-1,6-fucose-free sugar chains was87% and 8% in KM2760 and KM3060, respectively. Herein, the samples arereferred to as anti-CCR4 chimeric antibody (87%) and anti-CCR4 chimericantibody (8%).

The anti-CCR4 chimeric antibody (87%) and anti-CCR4 chimeric antibody(8%) were mixed at a ratio of anti-CCR4 chimeric antibody (87%):anti-CCR4 chimeric antibody (8%)=1:39, 16:67, 22:57, 32:47 or 42:37.Sugar chains of these samples were analyzed in accordance with themethod of Example 10(6). The ratio of α-1,6-fucose-free sugar chains was9%, 18%, 27%, 39% and 46%, respectively. Herein, these samples arereferred to as anti-CCR4 chimeric antibody (9%), anti-CCR4 chimericantibody (18%), anti-CCR4 chimeric antibody (27%), anti-CCR4 chimericantibody (39%) and anti-CCR4 chimeric antibody (46%).

Results of the sugar chain analysis of each of the samples are shown inFIG. 16. The ratio of α-1,6-fucose-free sugar chains was shown as anaverage value of the result of two sugar chain analyses.

6. Evaluation of Binding Activity to CCR4 Partial Peptide (ELISA)

Binding activity of the six kinds of the different anti-CCR4 chimericantibodies having a different α-1,6-fucose-free sugar chain prepared inthe item 5 of Example 8 to CCR4 partial peptide was measured inaccordance with the method described in the item 2 of Example 8.

As a result, as shown in FIG. 17, the six kinds of the anti-CCR4chimeric antibodies showed almost the same CCR4-binding activity, it wasfound that the ratio of α-1,6-fucose-free sugar chains does not haveinfluence on the antigen-binding activity of the antibody.

7. Evaluation of ADCC Activity on Human CCR4-High Expressing Cell Line

The ADCC activity of the anti-CCR4 chimeric antibodies against a humanCCR4-high expressing cell CCR4/EL-4 cell (WO 01/64754) was measured asfollows.

(1) Preparation of Target Cell Suspension

Cells (1.5×10⁶) of a human CCR4-expressing cell, CCR4/EL-4 cell,described in WO 01/64754 were prepared and a 5.55 MBq equivalent of aradioactive substance Na₂ ⁵¹CrO₄ was added thereto, followed by reactionat 37° C. for 1.5 hours to thereby label the cells with a radioisotope.After the reaction, the cells were washed three times by suspension in amedium and subsequent centrifugation, resuspended in the medium and thenincubated at 4° C. for 30 minutes in ice for spontaneous dissociation ofthe radioactive substance. After centrifugation, the cells were adjustedto give a density of 2×10⁵ cells/ml by adding 7.5 ml of the medium andused as a target cell suspension.

(2) Preparation of Human Effector Cell Suspension

From a healthy person, 60 ml of peripheral blood was collected, 0.6 mlof heparin sodium (manufactured by Shimizu Pharmaceutical) was addedthereto, followed by gently mixing. The mixture was centrifuged (800 g,20 minutes) to isolate a mononuclear cell layer using Lymphoprep(manufactured by AXIS SHIELD) in accordance with the manufacture'sinstructions. The cells were washed by centrifuging (1,400 rpm, 5minutes) three times using a medium and then re-suspended in the mediumto give a density of 5×10⁶ cells/ml and used as a human effector cellsuspension.

(3) Measurement of ADCC Activity

The target cell suspension prepared in the (1) was dispensed at 50 μl(1×10⁴ cells/well) into each well of a 96 well U-bottom plate(manufactured by Falcon). Next, 100 μl of the human effector cellsuspension prepared in the (2) was added thereto (5×10⁵ cells/well,ratio of the human effector cells to the target cells was 50:1).Furthermore, each of the anti-CCR4 chimeric antibodies was added theretoto give a final concentration of 0.0001 to 10 μg/ml, followed byreaction at 37° C. for 4 hours. After the reaction, the plate wascentrifuged and the amount of ⁵¹Cr in the supernatant was measured usinga γ-counter. An amount of the spontaneously dissociated ⁵¹Cr wascalculated by carrying out the same procedure using the medium aloneinstead of the human effector cell suspension and antibody solution, andmeasuring the amount of ⁵¹Cr in the supernatant. An amount of the totaldissociated ⁵¹Cr was calculated by carrying out the same procedure usinga 1 mol/L hydrochloric acid solution instead of the antibody solutionand human effector cell suspension, and measuring the amount of ⁵¹Cr inthe supernatant. The ADCC activity (%) was calculated based on equation(II).

FIGS. 18 and 19 show results of the measurement of ADCC activity of theanti-CCR4 chimeric antibodies having a different ratio ofα-1,6-fucose-free sugar chains at various concentrations (0.001 to 10μg/ml) using effector cells of two healthy donors (A and B),respectively. As shown in FIGS. 18 and 19, the ADCC activity of theanti-CCR4 chimeric antibodies showed a tendency to increase inproportion to the ratio of α-1,6-fucose-free sugar chains at eachantibody concentration. The ADCC activity decreases when the antibodyconcentration is low. At an antibody concentration of 0.01 μg/ml, theantibody in which the α-1,6-fucose-free sugar chains is 27%, 39% or 46%showed almost the same potent ADCC activity but the ADCC activity waslow in the antibody in which the ratio of α-1,6-fucose-free sugar chainsis less than 20%. The results were the same as the case when theeffector cell donor was changed.

Example 9 Determination of Transcription Product ofα-1,6-Fucosyltransferase Gene in Host Cell Line

(1) Preparation of Single-Stranded cDNA from Various Cell Lines

Single-stranded cDNA samples were prepared from dihydrofolate reductasegene (dhfr)-deleted CHO/DG44 cells derived from Chinese hamster ovaryand rat myeloma YB2/0 cells by the following procedure.

The CHO/DG44 cells were suspended in IMDM medium (manufactured by LifeTechnologies) supplemented with 10% fetal bovine serum (manufactured byLife Technologies) and 1× concentration HT supplement (manufactured byLife Technologies), and 15 ml of the suspension was inoculated into T75flask for adhesion cell culture use (manufactured by Greiner) at adensity of 2×10⁵ cells/ml. Also, the YB2/0 cells were suspended in RPMI1640 medium (manufactured by Life Technologies) supplemented with 10%fetal bovine serum (manufactured by Life Technologies) and 4 mmol/lL-GLN (manufactured by Life Technologies), and 15 ml of the suspensionwas inoculated into T75 flask for suspension cell culture (manufacturedby Greiner) at a density of 2×10⁵ cells/ml. They were cultured at 37° C.in a 5% CO₂ incubator, and 1×10⁷ of respective host cells were recoveredon the 1st, 2nd, 3rd, 4th and 5th days of the culturing to extract totalRNA using RNAeasy (manufactured by QIAGEN) in accordance with themanufacture's instructions.

The total RNA was dissolved in 45 μl of sterile water, 1 μl of RQ1RNase-Free DNase (manufactured by Promega), 5 μl of the attached10×DNase buffer and 0.5 μl of RNasin Ribonuclease Inhibitor(manufactured by Promega) were added thereto, followed by reaction at37° C. for 30 minutes to degrade genome DNA contaminated in the sample.After the reaction, the total RNA was purified again using RNAeasy(manufactured by QIAGEN) and dissolved in 50 μl of sterile water.

In a 20 μl of the reaction mixture using oligo(dT) as a primer,single-stranded cDNA was synthesized from 3 μg of each of the obtainedtotal RNA samples by reverse transcription reaction using SUPERSCRIPT™Preamplification System for First Strand cDNA Synthesis (manufactured byLife Technologies) and in accordance with the manufacture'sinstructions. A 1× concentration solution of the reaction solution wasused for the cloning of α-1,6-fucosyltransferase (hereinafter referredsometimes to as “FUT8”) and β-actin derived from respective host cells,and 50 folds-diluted aqueous solution of the reaction solution for thedetermination of each gene transcription amount by competitive PCR, andthe solutions were stored at −80° C. until use.

(2) Preparation of cDNA Partial Fragments of Chinese Hamster FUT8 andRat FUT8

Each cDNA partial fragment of Chinese hamster FUT8 and rat FUT8 wasprepared by the following procedure (FIG. 20).

First, primers (shown in SEQ ID NOs:4 and 5) specific for nucleotidesequences common to human FUT8 cDNA [J. Biochem., 121, 626 (1997)] andswine FUT8 cDNA [J. Biol. Chem., 271, 27810 (1995)] were designed.

Next, 25 μl of a reaction solution [ExTaq buffer (manufactured by TakaraShuzo), 0.2 mmol/l dNTPs and 0.5 μmol/l gene-specific primers (SEQ IDNOs:4 and 5)] containing 1 μl of each of the cDNA prepared from CHO celland cDNA prepared from YB2/0 cell, both obtained in the item (1) 2 daysafter culturing, and polymerase chain reaction (PCR) was carried outusing a DNA polymerase ExTaq (manufactured by Takara Shuzo). The PCR wascarried out by heating at 94° C. for 1 minute, subsequent 30 cycles ofheating at 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 2minutes as one cycle, and final heating at 72° C. for 10 minutes.

After the PCR, the reaction solution was subjected to 0.8% agarose gelelectrophoresis, and a specific amplified fragment of 979 bp waspurified using GENECLEAN Spin Kit (manufactured by BIO 101) and elutedwith 10 μl of sterile water (hereinafter, the method was used for thepurification of DNA fragments from agarose gel). Into a plasmid pCR2.1,4 μl of the amplified fragment was employed to insert in accordance withthe manufacture's instructions of TOPO TA Cloning Kit (manufactured byInvitrogen), and E. coli XL1-Blue was transformed with the reactionsolution by the method of Cohen et al. [Proc. Natl. Acad. Sci. USA, 69,2110 (1972)] (hereinafter, the method was used for the transformation ofE. coli). Plasmid DNA samples were isolated in accordance with a knownmethod [Nucleic Acids Research, 7, 1513 (1979)] (hereinafter, the methodwas used for the isolation of plasmid) from cDNA-inserted 6 clones amongthe obtained kanamycin-resistant colonies.

The nucleotide sequence of each cDNA inserted into the plasmid wasdetermined using DNA Sequencer 377 (manufactured by Parkin Elmer) andBigDye Terminator Cycle Sequencing FS Ready Reaction kit (manufacturedby Parkin Elmer) in accordance with the method of the manufacture'sinstructions. It was confirmed that all of the inserted cDNAs of whichsequences were determined by the method encode the open reading frame(ORF) partial sequences of Chinese hamster FUT8 or rat FUT8 (shown inSEQ ID NOs:6 and 7). Among these, plasmid DNA samples containingabsolutely no reading error by the PCR in the sequences were selected.Herein, these plasmids are referred to as CHFUT8-pCR2.1 andYBFUT8-pCR2.1.

(3) Preparation of Chinese Hamster β-Actin and Rat β-Actin cDNA

Chinese hamster β-actin and rat β-actin cDNA were prepared by thefollowing procedure (FIG. 21).

First, a forward primer specific for a common sequence containingtranslation initiation codon (shown in SEQ ID NO:8) and reverse primersspecific for respective sequences containing translation terminationcodon (shown in SEQ ID NOs:9 and 10) were designed from Chinese hamsterβ-actin genomic sequence (GenBank, U20114) and rat β-actin genomicsequence [Nucleic Acids Research, 11, 1759 (1983).

Next, 25 μl of a reaction solution [KOD buffer #1 (manufactured byToyobo), 0.2 mmol/l dNTPs, 1 mmol/l MgCl₂, 0.4 μmol/l gene-specificprimers (SEQ ID NOs:8 and 9, or SEQ ID NOs:8 and 10) and 5% DMSO]containing 1 μl of each of the cDNA prepared from CHO cell and cDNAprepared from YB2/0 cell, both obtained in the item (1) 2 days afterculturing was prepared, and PCR was carried out using a DNA polymeraseKOD (manufactured by Toyobo). The PCR was carried out by heating at 94°C. for 1 minute and subsequent cycles of heating at 98° C. for 15seconds, 65° C. for 2 seconds and 74° C. for 30 seconds as one cycle.

After the PCR, the reaction solution was subjected to 0.8% agarose gelelectrophoresis, and a specific amplified fragment of 1128 bp waspurified. The DNA fragment was subjected to DNA 5′-terminalphosphorylation using MEGALABEL (manufactured by Takara Shuzo) inaccordance with the manufacture's instructions. The DNA fragment wasrecovered from the reaction solution using ethanol precipitation methodand dissolved in 10 μl of sterile water.

Separately, 3 μg of a plasmid pBluescript II KS(+) (manufactured byStratagene) was dissolved in 35 μl of NEBuffer 2 (manufactured by NewEngland Biolabs), and 16 units of a restriction enzyme EcoRV(manufactured by Takara Shuzo) were added thereto for digestion reactionat 37° C. for 3 hours. To the reaction solution, 35 μl of 1 mol/lTris-HCl buffer (pH 8.0) and 3.5 μl of E. coli C15-derived alkalinephosphatase (manufactured by Takara Shuzo) were added thereto, followedby reaction at 65° C. for 30 minutes to thereby dephosphorylate the DNAterminus. The reaction solution was extracted with phenol/chloroform,followed by ethanol precipitation, and the recovered DNA fragment wasdissolved in 100 μl of sterile water.

Each 4 μl of the amplified fragment prepared from Chinese hamster cDNAor the amplified fragment (1192 bp) prepared from rat cDNA was mixedwith 1 μl of the EcoRV-EcoRV fragment (about 3.0 Kb) prepared fromplasmid pBluescript II KS(+) and 5 μl of Ligation High (manufactured byToyobo) for ligation reaction at 16° C. for 30 minutes. Using thereaction solution, E. coli XL1-Blue was transformed, and plasmid DNAsamples were isolated respectively in accordance with a known methodfrom the obtained ampicillin-resistant clones.

The nucleotide sequence of each cDNA inserted into the plasmid wasdetermined using DNA Sequencer 377 (manufactured by Parkin Elmer) andBigDye Terminator Cycle Sequencing FS Ready Reaction kit (manufacturedby Parkin Elmer) in accordance with the method of the manufacture'sinstructions. It was confirmed that all of the inserted cDNAs of whichsequences were determined by the method encode the ORF full sequences ofChinese hamster β-actin or rat β-actin. Among these, plasmid DNA samplescontaining absolutely no reading error of bases by the PCR in thesequences were selected. Herein, the plasmids are called CHAc-pBS andYBAc-pBS.

(4) Preparation of FUT8 Standard and Internal Control

In order to measure a transcription level of FUT8 gene mRNA in eachcell, CHFT8-pCR2.1 or YBFT8-pCR2.1, as plasmids in which cDNA partialfragments prepared in the item (2) from Chinese hamster FUT8 or rat FUT8were inserted into pCR2.1, respectively, were digested with arestriction enzyme EcoRI, and the obtained linear DNAs were used as thestandards for the preparation of a calibration curve. CHFT8d-pCR2.1 andYBFT8d-pCR2.1, which were obtained from the CHFT8-pCR2.1 andYBFT8-pCR2.1, by deleting 203 bp between ScaI and HindIII, an innernucleotide sequence of Chinese hamster FUT8 and rat FUT8, respectively,were digested with a restriction enzyme EcoRI, and the obtained linearDNAs were used as the internal standards for FUT8 amount determination.Details thereof are described below.

Chinese hamster FUT8 and rat FUT8 standards were prepared as follows. In40 μl of NEBuffer 2 (manufactured by New England Biolabs), 2 μg of theplasmid CHFT8-pCR2.1 was dissolved, 24 units of a restriction enzymeEcoRI (manufactured by Takara Shuzo) were added thereto, followed bydigestion reaction at 37° C. for 3 hours. Separately, 2 μg of theplasmid YBFT8-pCR2.1 was dissolved in 40 μl of NEBuffer 2 (manufacturedby New England Biolabs), and 24 units of a restriction enzyme EcoRI(manufactured by Takara Shuzo) were added thereto, followed by digestionreaction at 37° C. for 3 hours. By subjecting a portion of each of thereaction solutions to 0.8% agarose gel electrophoresis, it was confirmedthat an EcoRI-EcoRI fragment (about 1 Kb) containing each of cDNApartial fragments of Chinese hamster FUT8 and rat FUT8 was separatedfrom the plasmids CHFT8-pCR2.1 and YBFT8-pCR2.1 by the restrictionenzyme digestion reactions. Each of the reaction solutions was dilutedwith 1 μg/ml of baker's yeast t-RNA (manufactured by SIGMA) to give aconcentration of 0.02 fg/μl, 0.2 fg/μl, 1 fg/μl, 2 fg/μl, 10 fg/μl, 20fg/μl and 100 fg/μl and used as the Chinese hamster FUT8 and rat FUT8standards.

Internal standards of Chinese hamster FUT8 and rat FUT8 were prepared asfollows (FIG. 22). A reaction solution [KOD buffer #1 (manufactured byToyobo), 0.2 mmol/l dNTPs, 1 mmol/l MgCl₂, 0.4 mmol/l gene-specificprimers (SEQ ID NOs:11 and 12) and 5% DMSO] containing 5 ng ofCHFT8-pCR2.1 or YBFT8-pCR2.1 was prepared, and PCR was carried out usinga DNA polymerase KOD (manufactured by Toyobo). The PCR was carried outby heating at 94° C. for 4 minutes and subsequent 25 cycles of heatingat 98° C. for 15 seconds, 65° C. for 2 seconds and 74° C. for 30 secondsas one cycle. After the PCR, the reaction solution was subjected to 0.8%agarose gel electrophoresis, and a specific amplified fragment of about4.7 Kb was purified. The DNA 5′-terminal was phosphorylated usingMEGALABEL (manufactured by Takara Shuzo) in accordance with themanufacture's instructions, and then the DNA fragment was recovered fromthe reaction solution by ethanol precipitation and dissolved in 50 μl ofsterile water. The obtained DNA fragment (5 μl, about 4.7 kb) and 5 μlof Ligation High (manufactured by Toyobo) were mixed, followed byself-cyclization reaction at 16° C. for 30 minutes.

Using the reaction solution, E. coli DH5α was transformed, and plasmidDNA samples were isolated in accordance with a known method from theobtained ampicillin-resistant clones. The nucleotide sequence of eachplasmid DNA was determined using DNA Sequencer 377 (manufactured byParkin Elmer) and BigDye Terminator Cycle Sequencing FS Ready Reactionkit (manufactured by Parkin Elmer), and it was confirmed that a 203 bpinner nucleotide sequence between ScaI and HindIII of Chinese hamsterFUT8 or rat FUT8 was deleted. The obtained plasmids are referred to asCHFT8d-pCR2.1 or YBFT8d-pCR2.1, respectively.

Next, 2 μg of the plasmid CHFT8d-pCR2.1 was dissolved in 40 μl ofNEBuffer 2 (manufactured by New England Biolabs), and 24 units of arestriction enzyme EcoRI (manufactured by Takara Shuzo) were addedthereto, followed by digestion reaction at 37° C. for 3 hours.Separately, 2 μg of the plasmid YBFT8d-pCR2.1 was dissolved in 40 μl ofNEBuffer 2 (manufactured by New England Biolabs), and 24 units of arestriction enzyme EcoRI (manufactured by Takara Shuzo) were addedthereto, followed by digestion reaction at 37° C. for 3 hours. A portionof each of the reaction solutions was subjected to 0.8% agarose gelelectrophoresis, and it was confirmed that an EcoRI-EcoRI fragment(about 800 bp) containing a fragment from which 203 bp of the innernucleotide sequences of Chinese hamster FUT8 or rat FUT8 partialfragments was deleted was separated from the plasmids CHFT8d-pCR2.1 orYBFT8d-pCR2.1 by the restriction enzyme digestion reactions. Dilutionsof 2 fg/μl were prepared from the reaction solutions using 1 μg/mlbaker's yeast t-RNA (manufactured by SIGMA) and used as the Chinesehamster FUT8 or rat FUT8 internal controls.

(5) Preparation of β-Actin Standard and Internal Control

In order to measure the transcription amount of β-actin gene mRNA invarious host cells, CHAc-pBS and YBAc-pBS, as plasmids in which the ORFfull length of each cDNA of Chinese hamster β-actin and rat β-actinprepared in the item (3) was inserted into pBluescript II KS(+),respectively, were digested with restriction enzymes HindIII and PstIand restriction enzymes HindIII and KpnI, respectively, and the digestedlinear DNAs were used as the standards for the preparation of acalibration curve. CHAcd-pBS and YBAcd-pBS which were obtained from theCHAc-pBS and YBAc-pBS by deleting 180 bp between DraIII and DraIII of aninner nucleotide sequence of Chinese hamster β-actin and rat β-actinwere digested with restriction enzymes HindIII and PstI and restrictionenzymes HindIII and KpnI, respectively, and the digested linear DNAswere used as the internal standards for β-actin amount determination.Details thereof are described below.

Chinese hamster β-actin and rat β-actin standards were prepared asfollows. In 40 μl of NEBuffer 2 (manufactured by New England Biolabs), 2μg of the plasmid CHAc-pBS was dissolved, and 25 units of a restrictionenzyme HindIII (manufactured by Takara Shuzo) and 20 units of PstI(manufactured by Takara Shuzo) were added thereto, followed by digestionreaction at 37° C. for 3 hours. Separately, 2 μg of the plasmid YBAc-pBSwas dissolved in 40 μl of NEBuffer 2 (manufactured by New EnglandBiolabs), and 25 units of a restriction enzyme HindIII (manufactured byTakara Shuzo) and 25 units of KpnI (manufactured by Takara Shuzo) wereadded thereto, followed by digestion reaction at 37° C. for 3 hours. Aportion of each of the reaction solutions was subjected to 0.8% agarosegel electrophoresis, and it was confirmed that a HindIII-PstI fragmentand a HindIII-KpnI fragment (about 1.2 Kb) containing the full lengthORF of each cDNA of Chinese hamster β-actin and rat β-actin wereseparated from the plasmids CHAc-pBS and YBAc-pBS by the restrictionenzyme digestion reactions. Each of the reaction solutions was dilutedwith 1 μg/ml baker's yeast t-RNA (manufactured by SIGMA) to give aconcentration 2 pg/μl, 1 pg/μl, 200 fg/μl, 100 fg/μl and 20 fg/μl andused as the Chinese hamster β-actin and or β-actin standards.

Chinese hamster β-actin and rat β-actin internal standards were preparedas follows (FIG. 23). In 100 μl of NEBuffer 3 (manufactured by NewEngland Biolabs) containing 100 ng/μl of BSA (manufactured by NewEngland Biolabs), 2 μg of CHAc-pBS was dissolved, and 10 units of arestriction enzyme DraIII (manufactured by New England Biolabs) wereadded thereto, followed by digestion reaction at 37° C. for 3 hours. DNAfragments were recovered from the reaction solution by ethanolprecipitation and the DNA termini were changed to blunt ends using DNABlunting Kit (manufactured by Takara Shuzo) in accordance with themanufacture's instructions, and then the reaction solution was dividedinto two equal parts. First, to one part of the reaction solution, 35 μlof 1 mol/l Tris-HCl buffer (pH 8.0) and 3.5 of E. coli C15-derivedalkaline phosphatase (manufactured by Takara Shuzo) were added thereto,followed by reaction at 65° C. for 30 minutes for dephosphorylating theDNA termini. The DNA fragment was recovered by carrying outdephosphorylation treatment, phenol/chloroform extraction treatment andethanol precipitation treatment and then dissolved in 10 μl of sterilewater. The remaining part of the reaction solution was subjected to 0.8%agarose gel electrophoresis to purify a DNA fragment of about 1.1 Kbcontaining the ORF partial fragment of Chinese hamster β-actin.

The dephosphorylated DraIII-DraIII fragment (4.5 μl), 4.5 μl of theDraIII-DraIII fragment of about 1.1 Kb and 5 μl of Ligation High(manufactured by Toyobo) were mixed, followed by ligation reaction at16° C. for 30 minutes. Using the reaction solution, E. coli DH5α wastransformed, and plasmid DNAs were isolated in accordance with a knownmethod from the obtained ampicillin-resistant clones. The nucleotidesequence of each plasmid DNA was determined using DNA Sequencer 377(manufactured by Parkin Elmer) and BigDye Terminator Cycle Sequencing FSReady Reaction kit (manufactured by Parkin Elmer), and it was confirmedthat a Chinese hamster β-actin DraIII-DraIII 180 bp inserted into theplasmid was deleted. The plasmid is referred to as CHAcd-pBS.

Also, a plasmid in which rat β-actin DraIII-DraIII 180 bp was deletedwas prepared via the same steps of CHAcd-pBS. The plasmid is referred toas YBAcd-pBS.

Next, 2 μg of the plasmid CHAcd-pBS was dissolved in 40 μl of NEBuffer 2(manufactured by New England Biolabs), and 25 units of a restrictionenzyme HindIII (manufactured by Takara Shuzo) and 20 units of PstI(manufactured by Takara Shuzo) were added thereto, followed by digestionreaction at 37° C. for 3 hours. Separately, 2 μg of the plasmidYBAcd-pBS was dissolved in 40 μl of NEBuffer 2 (manufactured by NewEngland Biolabs), and 25 units of a restriction enzyme HindIII(manufactured by Takara Shuzo) and 24 units of KpnI (manufactured byTakara Shuzo) were added thereto, followed by digestion reaction at 37°C. for 3 hours. A portion of each of the reaction solutions wassubjected to 0.8% agarose gel electrophoresis, and it was confirmed thatan HindIII-PstI fragment and HindIII-KpnI fragment (about 1.0 Kb)containing a fragment in which 180 bp of the inner nucleotide sequenceof the ORF full length of each cDNA of Chinese hamster β-actin and ratβ-actin was deleted were separated from the plasmids CHAcd-pBS andYBAcd-pBS by the restriction enzyme digestion reactions. Dilutions of200 fg/μl were prepared from the reaction solutions using 1 μg/mlbaker's yeast t-RNA

(manufactured by SIGMA) and used as the Chinese hamster β-actin and ratβ-actin internal controls.

(6) Determination of Transcription Amount by Competitive PCR

Competitive PCR was carried out using the FUT8 internal control DNAprepared in the item (4) and the host cell-derived cDNA obtained in theitem (1) as the templates, the determined value of the FUT8transcription product in the host cell line was calculated from therelative value of the amount of the amplified product derived from eachtemplate. On the other hand, since it is considered that the β-actingene is transcribed continuously in each cell and its transcriptionlevel is approximately the same between cells, transcription level ofthe β-actin gene was determined as a measure of the efficiency ofsynthesis reaction of cDNA in each host cell line. That is, the PCR wascarried out using the β-actin internal control DNA prepared in the item(5) and the host cell-derived cDNA obtained in the item (1) as thetemplates, the determined value of the β-actin transcription product inthe host cell line was calculated from the relative value of the amountof the amplified product derived from each template. Details thereof aredescribed below.

The FUT8 transcription product was determined by the followingprocedure. First, a set of sequence-specific primers (shown in SEQ IDNOs:13 and 14) common to the inner sequences of the ORF partialsequences of Chinese hamster FUT8 and rat FUT8 obtained in the item (2)were designed.

Next, PCR was carried out using a DNA polymerase ExTaq (manufactured byTakara Shuzo) in 20 μl in total volume of a reaction solution [ExTaqbuffer (manufactured by Takara Shuzo), 0.2 mmol/l dNTPs, 0.5 mmol/lgene-specific primers (SEQ ID NOs:13 and 14) and 5% DMSO] containing 5μl of 50 folds-diluted cDNA solution prepared from each of respectivehost cell line in the item (1) and 5 μl (10 fg) of the plasmid forinternal control. The PCR was carried out by heating at 94° C. for 3minutes and subsequent 32 cycles of heating at 94° C. for 1 minute, 60°C. for 1 minute and 72° C. for 1 minute as one cycle.

Also, PCR was carried out in a series of reaction in which 5 μl (0.1 fg,1 fg, 5 fg, 10 fg, 50 fg, 100 fg, 500 fg or 1 pg) of the FUT8 standardplasmid obtained in the item (4) was added instead of the each host cellline-derived cDNA, and used in the preparation of a calibration curvefor the FUT8 transcription level.

The β-actin transcription product was determined by the followingprocedure. First, two sets of respective gene-specific primers common tothe inner sequences of the ORF full lengths of Chinese hamster β-actinand rat β-actin obtained in the item (3) were designed (the former areshown in SEQ ID NOs:15 and 16, and the latter are shown in SEQ ID NOs:17and 18).

Next, PCR was carried out using a DNA polymerase ExTaq (manufactured byTakara Shuzo) in 20 μl in total volume of a reaction solution [ExTaqbuffer (manufactured by Takara Shuzo), 0.2 mmol/l dNTPs, 0.5 mmol/lgene-specific primers (SEQ ID NOs:15 and 16, or SEQ ID NOs:17 and 18)and 5% DMSO] containing 5 μl of 50 folds-diluted cDNA solution preparedfrom respective host cell line in the item (1) and 5 μl (1 pg) of theplasmid for internal control. The PCR was carried out by heating at 94°C. for 3 minutes and subsequent 17 cycles of heating at 94° C. for 30seconds, 65° C. for 1 minute and 72° C. for 2 minutes as one cycle.

Also, PCR was carried out in a series of reaction in which 5 μl (10 pg,5 pg, 1 pg, 500 fg or 100 fg) of the β-actin standard plasmid obtainedin the item (5) was added instead of the each host cell line-derivedcDNA, and used in the preparation of a calibration curve for the β-actintranscription level.

TABLE 3 Size (bp) of PCR Target amplification product gene Primer set *Target Competitor FUT8 F: 5′-GTCCATGGTGATCCTGCAGTGTGG-3′ 638 431R: 5′-CACCAATGATATCTCCAGGTTCC-3′ β-ActinF: 5′-GATATCGCTGCGCTCGTTGTCGAC-3′ 789 609R: 5′-CAGGAAGGAAGGCTGGAAAAGAGC-3′ (Chinese hamster) β-ActinF: 5′-GATATCGCTGCGCTCGTCGTCGAC-3′ 789 609R: 5′-CAGGAAGGAAGGCTGGAAGAGAGC-3′ (Rat) * F: forward primer, R: reverseprimer

By carrying out PCR using the primer set described in Table 3, a DNAfragment having a size shown in the target column of Table 3 can beamplified from each gene transcription product and each standard, and aDNA fragment having a size shown in the competitor column of Table 3 canbe amplified from each internal control.

A 7 μl portion of each of the solutions after PCR was subjected to 1.75%agarose gel electrophoresis, and then the gel was stained by soaking itfor 30 minutes in 1× concentration SYBR Green I Nucleic Acid Gel Stain(manufactured by Molecular Probes). The amount of the amplified DNAfragment was measured by calculating luminescence intensity of eachamplified DNA using a fluoro-imager (FluorImager SI; manufactured byMolecular Dynamics).

The amount of an amplified product formed by PCR using a standardplasmid as the template was measured by the method, and a calibrationcurve was prepared by plotting the measured values against the amountsof the standard plasmid. Using the calibration curve, the amount of cDNAof a gene of interest in each cell line was calculated from the amountof the amplified product when each expression cell line-derived cDNA wasused as the template, and the amount was defined as the mRNAtranscription amount in each cell line.

The amount of the FUT8 transcription product in each host cell line whena rat FUT8 sequence was used in the standard and internal control isshown in FIG. 24. Throughout the culturing period, the CHO cell lineshowed a transcription amount 10 folds or higher than that of the YB2/0cell line. The tendency was also found when a Chinese hamster FUT8sequence was used in the standard and internal control.

Also, the FUT8 transcription amounts are shown in Table 4 as relativevalues to the amount of the β-actin transcription product. Throughoutthe culturing period, the FUT8 transcription amount in the YB2/0 cellline was around 0.1% of β-actin while it was 0.5% to 2% in the CHO cellline.

The results shows that the amount of the FUT8 transcription product inYB2/0 cell line was significantly smaller than that in the CHO cellline.

TABLE 4 Culture days Cell line 1st 2nd 3rd 4th 5th CHO 1.95 0.90 0.570.52 0.54 YB2/0 0.12 0.11 0.14 0.08 0.07

Example 10 Determination of Transcription Product ofα-1,6-Fucosyltransferase (FUT8) Gene in Anti-Ganglioside GD3 ChimericAntibody-Producing Cell Line

(1) Preparation of Single-Stranded cDNA from Various Antibody-ProducingCell Lines

Single-stranded cDNA was prepared from anti-ganglioside

GD3 chimeric antibody-producing cell lines DCHI01-20 and 61-33 asfollows. The DCHI01-20 is a transformant clone derived from the CHO/DG44cell described in item 2(2) of Example 1. Also, the 61-33 is a cloneobtained by carrying out serum-free adaptation of YB2/0-derivedtransformant cell 7-9-51 (FERM BP-6691, International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (AIST Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi,Ibaraki-ken 305-8566 Japan)) and then carrying out single cell isolationby two limiting dilution.

Cells of the DCHI01-20 were suspended in EXCELL 302 medium (manufacturedby JRH BIOSCIENCES) supplemented with 3 mmol/l L-GLN (manufactured byLife Technologies), 0.3% PLURONIC F-68 (manufactured by LifeTechnologies) and 0.5% fatty acid concentrate (manufactured by LifeTechnologies), and 15 ml of the suspension was inoculated into T75 flaskfor suspension cell culture use (manufactured by Greiner) at a densityof 2×10⁵ cells/ml. Also, cells of the 61-33 were suspended inHybridoma-SFM medium (manufactured by Life Technologies) supplementedwith 0.2% bovine serum albumin fraction V (manufactured by LifeTechnologies) (hereinafter referred to as “BSA”), and 15 ml of thesuspension was inoculated into T75 flask for suspension cell culture(manufactured by Greiner) at a density of 2×10⁵ cells/ml. They werecultured at 37° C. in a 5% CO₂ incubator, and 1, 2, 3, 4 and 5 daysafter culturing, 1×10⁷ of respective host cells were recovered toextract total RNA using RNAeasy (manufactured by QIAGEN) in accordancewith the manufacture's instructions.

The total RNA was dissolved in 45 μl of sterile water, and 1 μl of RQ1RNase-Free DNase (manufactured by Promega), 5 μl of the attached10×DNase buffer and 0.5 μl of RNasin Ribonuclease Inhibitor(manufactured by Promega) were added thereto, followed by reaction at37° C. for 30 minutes to degrade genome DNA contaminated in the sample.After the reaction, the total RNA was purified again using RNAeasy(manufactured by QIAGEN) and dissolved in 50 μl of sterile water.

In a 20 μl reaction mixture using oligo(dT) as a primer, single-strandedcDNA was synthesized from 3 μg of each of the obtained total RNA samplesby reverse transcription reaction using SUPERSCRIPT™ PreamplificationSystem for First Strand cDNA Synthesis (manufactured by LifeTechnologies) in accordance with the manufacture's instructions. Thereaction solution was diluted 50 folds with water and stored at −80° C.until use.

(2) Determination of Transcription Amounts of Each Gene by CompetitivePCR

The transcription amount of each of the genes on the cDNA derived fromthe antibody-producing cell line obtained in the item (1) was determinedby competitive PCR in accordance with Example 9(6).

The FUT8 gene-derived mRNA transcription amount in each of theantibody-producing cell lines was determined by the following procedure.

CHFT8-pCR2.1 and YBFT8-pCR2.1, as plasmids in which cDNA partialfragments prepared in Example 9(2) from Chinese hamster FUT8 and ratFUT8, respectively, were inserted into pCR2.1, were digested with arestriction enzyme EcoRI, and the obtained linear DNAs were used as thestandards in the preparation of a calibration curve for determining theFUT8 transcription amount.

CHFT8d-pCR2.1 and YBFT8d-pCR2.1, which were obtained by deleting 203 bpbetween ScaI and HindIII of an inner nucleotide sequence of Chinesehamster FUT8 and rat FUT8, respectively, in Example 9(4) were digestedwith a restriction enzyme EcoRI, and the obtained linear DNAs were usedas the internal standards for FUT8 amount determination.

PCR was carried out using a DNA polymerase ExTaq (manufactured by TakaraShuzo) in 20 μl in total volume of a reaction solution [ExTaq buffer(manufactured by Takara Shuzo), 0.2 mmol/l dNTPs, 0.5 μmol/l FUT8gene-specific primers (SEQ ID NOs:13 and 14) and 5% DMSO] containing 5μl of 50 folds-diluted cDNA solution prepared from each of theantibody-producing cell line in the item (1) and 5 μl (10 fg) of theplasmid for internal control. The PCR was carried out by heating at 94°C. for 3 minutes and subsequent cycles of heating at 94° C. for 1minute, 60° C. for 1 minute and 72° C. for 1 minute as one cycle.

Also, PCR was carried out in a series of reaction in which 5 μl (0.1 fg,1 fg, 5 fg, 10 fg, 50 fg, 100 fg, 500 fg or 1 pg) of the FUT8 standardplasmid was added instead of the each antibody-producing cellline-derived cDNA, and used in the preparation of a calibration curvefor the FUT8 transcription amount. In this case, 1 μg/ml of a baker'syeast t-RNA (manufactured by SIGMA) was used for the dilution of thestandard plasmid.

On the other hand, since it is considered that the β-actin gene istranscribed constantly in each cell and its transcription amount isapproximately the same between cells, the transcription amount of theβ-actin gene was determined as an index of the efficiency of synthesisreaction of cDNA in each antibody-producing cell line.

CHAc-pBS and YBAc-pBS as plasmids in which the ORF full length of eachcDNA of Chinese hamster β-actin and rat β-actin prepared in Example 9(3)were inserted into pBluescript II KS(+), respectively, were digestedwith restriction enzymes HindIII and KpnI, and the obtained linear DNAsamples were used as the standards in the preparation of a calibrationcurve for determining the β-actin transcription amount.

CHAcd-pBS and YBAcd-pBS which were obtained by deleting 180 bp betweenDraI and DraI of an inner nucleotide sequence of Chinese hamster β-actinand rat β-actin, respectively in Example 9(5), were digested withrestriction enzymes HindIII and KpnI, and the obtained linear DNAs wereused as the internal standards for β-actin determination.

PCR was carried out using a DNA polymerase ExTaq (manufactured by TakaraShuzo) in 20 μl in total volume of a reaction solution [ExTaq buffer(manufactured by Takara Shuzo), 0.2 mmol/l dNTPs, 0.5 μmol/lβ-actin-specific primers (SEQ ID NOs:17 and 18) and 5% DMSO] containing5 μl of 50 folds-diluted cDNA solution prepared from each of theantibody-producing cell lines and 5 μl (1 pg) of the plasmid forinternal control. The PCR was carried out by heating at 94° C. for 3minutes and subsequent 17 cycles of heating at 94° C. for 30 seconds,65° C. for 1 minute and 72° C. for 2 minutes as one cycle. Also, PCR wascarried out in a series of reaction in which 10 pg, 5 pg, 1 pg, 500 fgor 100 fg of the β-actin standard plasmid was added instead of the eachantibody-producing cell line-derived cDNA, and used in the preparationof a calibration curve for the β-actin transcription amount. In thiscase, 1 μg/ml of a baker's yeast t-RNA (manufactured by SIGMA) was usedfor the dilution of standard plasmid.

By PCR using the primer set described in Table 3, a DNA fragment havinga size shown in the target column of Table 3 can be amplified from eachgene transcription product and each standard, and a DNA fragment havinga size shown in the competitor column of Table 3 can be amplified fromeach internal control.

A 7 μl portion of each of the solutions after PCR was subjected to 1.75%agarose gel electrophoresis, and then the gel was stained by soaking itfor 30 minutes in 1× concentration SYBR Green I Nucleic Acid Gel Stain(manufactured by Molecular Probes). The amount of the amplified DNAfragment was measured by calculating luminescence intensity of eachamplified DNA using a fluoro-imager (FluorImager SI; manufactured byMolecular Dynamics).

The amount of the amplified product formed by PCR which used a standardplasmid as the template was measured by the method, and a calibrationcurve was prepared by plotting the measured values against the amountsof the standard plasmid. Using the calibration curve, the amount of cDNAof a gene of interest in each cell line was calculated from the amountof the amplified product when each antibody-producing cell line-derivedcDNA was used as the template, and the value was defined as the mRNAtranscription amount in each cell line.

The FUT8 transcription amounts are shown in Table 5 as relative valuesto the amount of the β-actin transcription product. Throughout theculturing period, the FUT8 transcription amount in the YB2/0cell-derived antibody-producing 61-33 was 0.3% or less of β-actin whileit was 0.7% to 1.5% in the CHO cell-derived antibody-producing cell.

The results shows that the amount of the FUT8 transcription product inthe YB2/0 cell-derived antibody-producing cell line was significantlyless than that in the antibody-producing cell line derived from the CHOcell.

TABLE 5 Culture days Cell line 1st 2nd 3rd 4th 5th DCHI01-20 0.75 0.730.99 1.31 1.36 61-33 0.16 0.19 0.24 0.30 <0.10

Example 11 Preparation of Mouse α-1,6-Fucosyltransferase (FUT8) GeneOver-Expressing Cell Line (1) Construction of Mouseα-1,6-Fucosyltransferase (FUT8) Expression Plasmid

Total RNA was extracted from 1×10⁷ cells of a mouse myeloma NS0 cell(RCB0213, Cell Bank at The Institute of Physical and Chemical Research)subcultured using IMDM medium (manufactured by Life Technologies)containing 10% fetal bovine serum (manufactured by Life Technologies),using RNAeasy (manufactured by QIAGEN) in accordance with themanufacture's instructions. The total RNA was dissolved in 45 μl ofsterile water, and 1 μl of RQ1 RNase-Free DNase (manufactured byPromega), 5 μl of the attached 10×DNase buffer and 0.5 μl of RNasinRibonuclease Inhibitor (manufactured by Promega) were added thereto,followed by reaction at 37° C. for 30 minutes to degrade genome DNAcontaminated in the sample. After the reaction, the total RNA waspurified again using RNAeasy (manufactured by QIAGEN) and dissolved in50 μl of sterile water. In a 20 μl reaction mixture using oligo(dT) as aprimer, single-stranded cDNA was synthesized from 3 μg of the obtainedtotal RNA by reverse transcription reaction using SUPERSCRIPT™Preamplification System for First Strand cDNA Synthesis (manufactured byLife Technologies) in accordance with the manufacture's instructions.

Mouse FUT8 cDNA was prepared by the following procedure (FIG. 25).

First, a forward primer specific for a sequence containing a translationinitiation codon (shown in SEQ ID NO:19) and a reverse primer specificfor a sequence containing translation termination codon (shown in SEQ IDNO:20) were designed from a mouse FUT8 cDNA sequence (GenBank,AB025198).

Next, 25 μl of a reaction solution [ExTaq buffer (manufactured by TakaraShuzo), 0.2 mmol/l dNTPs, 4% DMSO and 0.5 μmol/l specific primers (SEQID NO:19 and SEQ ID NO:20)] containing 1 μl of the NSO cell-derived cDNAwas prepared, and PCR was carried out using a DNA polymerase ExTaq(manufactured by Takara Shuzo). The PCR was carried out by heating at94° C. for 1 minute, subsequent 30 cycles of heating at 94° C. for 30seconds, 55° C. for 30 seconds and 72° C. for 2 minutes as one cycle,and final heating at 72° C. for 10 minutes.

After the PCR, the reaction solution was subjected to 0.8% agarose gelelectrophoresis, and a specific amplified fragment of 1728 bp waspurified. Into a plasmid pCR2.1, 4 μl of the DNA fragment was employedto insert in accordance with the manufacturer's instructions attached toTOPO TA Cloning Kit (manufactured by Invitrogen), and E. coli DH5α wastransformed with the reaction solution. Plasmid DNAs were isolated inaccordance with a known method from cDNA-inserted 6 clones among theobtained kanamycin-resistant colonies.

The nucleotide sequence of each cDNA inserted into the plasmid wasdetermined using DNA Sequencer 377 (manufactured by Parkin Elmer) andBigDye Terminator Cycle Sequencing FS Ready Reaction kit (manufacturedby Parkin Elmer) in accordance with the method of the manufacture'sinstructions. It was confirmed that all of the inserted cDNAs of whichsequences were determined encode the ORF full sequence of mouse FUT8.Among these, a plasmid DNA containing absolutely no reading error ofbases by the PCR in the sequences were selected (its DNA sequence andamino acid sequence are shown in SEQ ID NOs:2 and 24, respectively).Also, inconsistency of 3 bases due to amino acid substitution was foundin the sequence when compared with the mouse FUT8 sequence registered onGenBank. Herein, the plasmid is referred to mfFUT8-pCR2.1.

Next, a plasmid pBSmfFUT8 containing the ORF full sequence of mouse FUT8was constructed as follows (FIG. 26). First, 1 μg of a plasmidpBluescript II KS(+) (manufactured by Stratagene) was dissolved in 35 μlof NEBuffer 2 (manufactured by New England Biolabs), and 20 units of arestriction enzyme EcoRI (manufactured by Takara Shuzo) were addedthereto, followed by digestion reaction at 37° C. for 2 hours. To thereaction solution, 35 μl of 1 mol/l Tris-HCl buffer (pH 8.0) and 3.5 μlof E. coli C15-derived alkaline phosphatase (manufactured by TakaraShuzo) were added, followed by reaction at 65° C. for 30 minutes fordephosphorylate the DNA termini. The reaction solution was extractedwith phenol/chloroform, followed by ethanol precipitated, and therecovered DNA fragment was dissolved in 10 μl of sterile water.

Separately, 1 μg of the plasmid mfFUT8-pCR2.1 was dissolved in 35 μl ofNEBuffer 2 (manufactured by New England Biolabs), and 20 units of arestriction enzyme EcoRI (manufactured by Takara Shuzo) were addedthereto, followed by digestion reaction at 37° C. for 2 hours. Thereaction solution was subjected to 0.8% agarose gel electrophoresis topurify a DNA fragment of about 1.7 Kb containing the ORF full sequenceof mouse FUT8 cDNA.

The obtained plasmid pBluescript II KS(+)-derived EcoRI-EcoRI fragment(1 μl, 2.9 Kb), 4 μl of the EcoRI-EcoRI fragment (1.7 Kb) prepared fromthe plasmid mfFUT8-pCR2.1 and 5 μl of Ligation High (manufactured byToyobo) were mixed, followed by ligation reaction at 16° C. for 30minutes. Using the reaction solution, E. coli DH5α was transformed, andplasmid DNAs were isolated in accordance with a known method from theobtained ampicillin-resistant clones. Herein, the plasmid is referred topBSmfFUT8.

Using the pBSmfFUT8 and pAGE249, a mouse FUT8 expression vectorpAGEmfFUT8 was constructed by the following procedure (FIG. 27). ThepAGE249 is a derivative of pAGE248 [J. Biol. Chem., 269, 14730 (1994)],as a vector in which an SphI-SphI fragment (2.7 Kb) containing adihydrofolate reductase gene (dhfr) expression unit was removed from thepAGE248.

In 50 μl of Universal Buffer H (manufactured by Takara Shuzo), 1 μg ofthe pAGE249 was dissolved, and 20 units of a restriction enzyme SalI(manufactured by New England Biolabs) were added thereto, followed bydigestion reaction at 37° C. for 2 hours. A DNA fragment was recoveredfrom the reaction solution by ethanol precipitation and dissolved in 35μl of NEBuffer 2 (manufactured by New England Biolabs), and 20 units ofa restriction enzyme BamHI (manufactured by New England Biolabs) wereadded thereto, followed by digestion reaction at 37° C. for 2 hours.After the digestion reaction, to the reaction solution, 35 μl of 1 mol/lTris-HCl buffer (pH 8.0) and 3.5 μl of E. coli C15-derived alkalinephosphatase (manufactured by Takara Shuzo) were added thereto, followedby reaction at 65° C. for 30 minutes to dephosphorylate the DNA termini.The reaction solution was extracted with phenol/chloroform extraction,followed by ethanol precipitation, and the recovered DNA fragment wasdissolved in 10 μl of sterile water.

Separately, 1 μg of pBSmfFUT8 was dissolved in 50 μl of Universal BufferH (manufactured by Takara Shuzo), and 20 units of a restriction enzymeSalI (manufactured by New England Biolabs) were added thereto, followedby digestion reaction at 37° C. for 2 hours. A DNA fragment wasrecovered from the reaction solution by ethanol precipitation anddissolved in 35 μl of NEBuffer 2 (manufactured by New England Biolabs),and 20 units of a restriction enzyme BamHI (manufactured by New EnglandBiolabs) were added thereto, followed by digestion reaction at 37° C.for 2 hours. After the digestion reaction, the solution was subjected to0.8% agarose gel electrophoresis to purify a DNA fragment of about 1.7Kb containing the ORF full sequence of mouse FUT8 cDNA.

The obtained plasmid pAGE249-derived BamHI-SalI fragment (1 μl, 6.5 Kb),4 μl of the BamHI-SalI fragment (1.7 Kb) prepared from the plasmidpBSmfFUT8 and 5 μl of Ligation High (manufactured by Toyobo) were mixed,followed by ligation reaction at 16° C. for 30 minutes. Using thereaction solution, E. coli DH5α was transformed, and a plasmid DNA wasisolated in accordance with a known method from the obtainedampicillin-resistant clones. Herein, the plasmid is referred topAGEmfFUT8.

(2) Preparation of Mouse α-1,6-Fucosyltransferase (FUT8) GeneOver-Expressing Cell Line

A stable FUT8 gene-expressing cell line was obtained by introducing themouse FUT8 expression vector pAGEmfFUT8 constructed in the item (1) into61-33. The 61-33 is a clone obtained by carrying out serum-freeadaptation of a transformant cell 7-9-51 (FERM BP-6691, InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology) derived from a YB2/0 cell highly producing ananti-ganglioside GD3 chimeric antibody, and then carrying out singlecell isolation by two limiting dilution.

The plasmid pAGEmfFUT8 was transferred into 61-33 by the followingprocedure in accordance with the electroporation [Cytotechnology, 3, 133(1990)]. First, 30 μg of the plasmid pAGEmfFUT8 was dissolved in 600 μlof NEBuffer 4 (manufactured by New England Biolabs), and 100 units of arestriction enzyme FspI (manufactured by New England Biolabs) were addedthereto, followed by digestion reaction at 37° C. for 2 hours to obtaina linear fragment. The reaction solution was subjected to ethanolprecipitation, and the recovered linear plasmid was made into a 1 μg/μlaqueous solution. Next, the 61-33 was suspended in a K-PBS buffer (137mol/l KCl, 2.7 mol/l NaCl, 8.1 mol/l Na₂HPO₄, 1.5 mol/l KH₂PO₄, 4.0mol/l MgCl₂) to give a density of 2×10⁷ cells/ml, and 200 μl of the cellsuspension (4×10⁶ cells) was mixed with 10 μl (10 μg) of the linearplasmid. The cell-DNA mixture was transferred into Gene Pulser Cuvette(inter-electrode distance, 2 mm) (manufactured by BIO-RAD) and thenelectroporation was carried out using a cell fusion apparatus GenePulser (manufactured by BIO-RAD) at 0.2 KV pulse voltage and 250 μFelectric capacity. The cell suspension was mixed with ml ofHybridoma-SFM medium (manufactured by Life Technologies) supplementedwith 5% fetal bovine dialyzed serum (manufactured by Life Technologies)and 0.2% BSA (manufactured by Life Technologies) and dispensed in 100 μlportions into a 96 well plate for suspension cell use (manufactured byGreiner). After culturing them at 37° C. for 24 hours in 5% CO₂, 50 μlof the culture supernatant was removed, and Hybridoma-SFM medium(manufactured by Life Technologies) supplemented with 0.5 mg/mlHygromycin B (manufactured by Wako Pure Chemical Industries), 5% fetalbovine dialyzed serum (manufactured by Life Technologies) and 0.2% BSA(manufactured by Life Technologies) was dispensed at 100 μl. They werecultured for 3 weeks while repeating the medium exchange step atintervals of 3 to 4 days, and 14 cell lines showing hygromycinresistance were obtained.

On the other hand, a negative control cell line was prepared byintroducing the plasmid pAGE249 as a parent vector of the pAGEmfFUT8into the 61-33. According to the above procedure, 10 μg of the plasmidpAGE249 converted into linear form with a restriction enzyme FspI wasintroduced into 4×10⁶ cells of the 61-33 using the electroporation. Thecells were mixed with 15 ml of Hybridoma-SFM medium (manufactured byLife Technologies) supplemented with 5% fetal bovine dialyzed serum(manufactured by Life Technologies) and 0.2% BSA (manufactured by LifeTechnologies), transferred into a T75 flask for suspension cell(manufactured by Greiner) and then cultured at 37° C. for 24 hours in 5%CO₂. After culturing them, a half of the culture supernatant (7.5 ml)was removed by centrifugation at 800 rpm for 4 minutes, and the cellswere suspended in 7.5 ml of Hybridoma-SFM medium (manufactured by LifeTechnologies) supplemented with 0.5 mg/ml Hygromycin B (manufactured byWako Pure Chemical Industries), 5% fetal bovine dialyzed serum(manufactured by Life Technologies) and 0.2% BSA (manufactured by LifeTechnologies) and transferred into the T75 flask for suspension cell(manufactured by Greiner). They were cultured for 3 weeks whilerepeating the medium exchange at intervals of 3 to 4 days, ahygromycin-resistant cell line was obtained.

(3) Analysis of Expression Level of α-1,6-Fucosyltransferase (FUT8) Genein Cell Lines Over-Expressing the Gene

Using 6 cell lines optionally selected from the 14 mouse FUT8-overexpressing cell lines prepared from 61-33 in the item (2) and thenegative control cell line, the FUT8 expression levels were comparedusing competitive RT-PCR.

Each of these over-expression cell lines was suspended in Hybridoma-SFMmedium (manufactured by Life Technologies) supplemented with 0.5 mg/mlHygromycin B (manufactured by Wako Pure Chemical Industries), 5% fetalbovine dialyzed serum (manufactured by Life Technologies) and 0.2% BSA(manufactured by Life Technologies) to give a density of 3×10⁵ cells/mland then transferred into a T75 flask for suspension cell culture use(manufactured by Greiner). After culturing them at 37° C. for 24 hoursin 5% CO₂, 1×10⁷ of intact cells were recovered to extract total RNAusing RNAeasy (manufactured by QIAGEN) in accordance with themanufacture's instructions. The total RNA was dissolved in 45 μl ofsterile water, and 0.5 U/μl of RQ1 RNase-Free DNase (manufactured byPromega), 5 μl of the attached 10×DNase buffer and 0.5 μl of RNasinRibonuclease Inhibitor (manufactured by Promega) were added thereto,followed by reaction at 37° C. for 30 minutes to degrade genome DNAcontaminated in the sample. After the reaction, the total RNA waspurified again using RNAeasy (manufactured by QIAGEN) and dissolved in50 μl of sterile water.

In a 20 μl reaction mixture using oligo(dT) as a primer, single-strandedcDNA was synthesized from 2.5 μg of the obtained total RNA by reversetranscription reaction using SUPERSCRIPT™ Preamplification System forFirst Strand cDNA Synthesis (manufactured by Life Technologies) inaccordance with the manufacture's instructions. The reaction solutionwas diluted 50 folds with water and the transcription amount of eachgene was determined by the competitive PCR in accordance with Example9(6).

The FUT8 gene-derived mRNA transcription amount in each expression cellline was determined by the following procedure.

YBFT8-pCR2.1, as a plasmid in which a cDNA partial fragment prepared inExample 9(2) from rat FUT8 was inserted into pCR2.1, was digested with arestriction enzyme EcoRI, and the obtained linear DNA was used as thestandard in the preparation of a calibration curve for determining theFUT8 transcription amount.

Among the YBFT8-pCR2.1 prepared in Example 9(4), YBFT8d-pCR2.1 obtainedby deleting 203 bp between ScaI and HindIII of an inner nucleotidesequence of rat FUT8 was digested with a restriction enzyme EcoRI, andthe obtained linear DNA was used as the internal control for FUT8determination.

PCR was carried out using a DNA polymerase ExTaq (manufactured by TakaraShuzo) in 20 μl in total volume of a reaction solution [ExTaq buffer(manufactured by Takara Shuzo), 0.2 mmol/l dNTPs, 0.5 mmol/l FUT8gene-specific primers (SEQ ID NOs:13 and 14) and 5% DMSO] containing 5μl of 50 folds-diluted cDNA solution prepared from respective expressioncell line in the above and 5 μl (10 fg) of the plasmid for internalcontrol. The PCR was carried out by heating at 94° C. for 3 minutes andsubsequent 32 cycles of heating at 94° C. for 1 minute, 60° C. for 1minute and 72° C. for 1 minute as one cycle.

Also, PCR was carried out in a series of reaction in which 5 μl (0.1 fg,1 fg, 5 fg, 10 fg, 50 fg, 100 fg, 500 fg or 1 pg) of the FUT8 standardplasmid was added instead of the each expression cell line-derived cDNA,and used in the preparation of a calibration curve for the FUT8transcription amount. In this case, 1 μg/ml baker's yeast t-RNA(manufactured by SIGMA) was used for the dilution of standard plasmid.

On the other hand, since it is considered that the β-actin gene istranscribed constantly in each cell and its transcription level isapproximately the same between cells, the transcription amount of theβ-actin gene was determined as an index of the efficiency of synthesisreaction of cDNA in each expression cell line.

YBAc-pBS, as a plasmid in which the ORF full sequence of cDNA of ratβ-actin was inserted into pBluescript II KS(+) prepared in Example 9(3),was digested with restriction enzymes HindIII and KpnI, and the obtainedlinear DNA was used as the standard in the preparation of a calibrationcurve for determining the β-actin gene transcription amount.

YBAcd-pBS obtained from the YBAc-pBS by deleting 180 bp between DraI andDraI of an inner nucleotide sequence of rat β-actin was digested withrestriction enzymes HindIII and KpnI, and the obtained linear DNA wasused as the internal standards for β-actin amount determination.

PCR was carried out using a DNA polymerase ExTaq (manufactured by TakaraShuzo) in 20 μl in total volume of a reaction solution [ExTaq buffer(manufactured by Takara Shuzo), 0.2 mmol/l dNTPs, 0.5 μmol/lβ-actin-specific primers (SEQ ID NOs:17 and 18) and 5% DMSO] containing5 μl of 50 folds-diluted cDNA solution prepared from each of theexpression cell lines and 5 μl (1 pg) of the plasmid for internalcontrol. The PCR was carried out by heating at 94° C. for 3 minutes andsubsequent 17 cycles of heating at 94° C. for 30 seconds, 65° C. for 1minute and 72° C. for 2 minutes as one cycle.

Also, PCR was carried out in a series of reaction in which 10 pg, 5 pg,1 pg, 500 fg or 100 fg of the β-actin standard plasmid was added insteadof the each expression cell line-derived cDNA, and used in thepreparation of a calibration curve for the β-actin transcription amount.In this case, 1 μg/ml baker's yeast t-RNA (manufactured by SIGMA) wasused for diluting the standard plasmid.

By carrying out PCR using the primer set described in Table 3, a DNAfragment having a size shown in the target column of Table 3 can beamplified from each gene transcription product and each standard, and aDNA fragment having a size shown in the competitor column of Table 3 canbe amplified from each internal control.

Each (7 μl) of the solutions after PCR was subjected to a 1.75% agarosegel electrophoresis, and then the gel was stained by soaking it for 30minutes in 1× concentration SYBR Green I Nucleic Acid Gel Stain(manufactured by Molecular Probes). By calculating luminescenceintensity of each amplified DNA fragment using a fluoro-imager(FluorImager SI; manufactured by Molecular Dynamics), the amount of theamplified DNA fragment was measured.

The amount of an amplified product formed by PCR using the standardplasmid as the template was measured by the method, and a calibrationcurve was prepared by plotting the measured values against the amountsof the standard plasmid. Using the calibration curve, the amount of cDNAof a gene of interest in each cell line was calculated from the amountof an amplified product when each expression cell line-derived cDNA wasused as the template, and the amount was defined as the mRNAtranscription amount in each cell line.

FIG. 28 shows the FUT8 transcription amounts as relative values to theamount of β-actin transcription product. Three cell lines mfFUT8-1,mfFUT8-2 and mfFUT8-4 and the pAGE249-introduced cell line were celllines having a relatively small FUT8 transcription amount, which wasequivalent to 0.3 to 10% of a β-actin transcription amount. On the otherhand, other three cell lines mfFUT8-3, mfFUT8-6 and mfFUT8-7 were celllines having a relatively large FUT8 transcription amount, which wasequivalent to 20 to 40% of a β-actin transcription amount.

(4) Purification of Antibody Produced by α-1,6-fucosyltransferase (FUT8)gene over-expressing cell line

Each of the six FUT8 gene over-expressing cell lines and one negativecontrol cell line obtained in the item (2) was suspended inHybridoma-SFM medium (manufactured by Life Technologies) supplementedwith 200 nmol/l MTX, 0.5 mg/ml Hygromycin B (manufactured by Wako PureChemical Industries) and 0.2% BSA (manufactured by Life Technologies) togive a density of 2×10⁵ cells/ml, and then 100 ml in total of thesuspension was inoculated into three T225 flasks for suspension cellculture use (manufactured by IWAKI). After culturing them at 37° C. for7 to 9 days in a 5% CO₂ incubator, the number of intact cells wascounted to confirm that their viability was almost the same (each 30% orless), and then each cell suspension was recovered. Each of the cellsuspensions was centrifuged at 3,000 rpm at 4° C. for 10 minutes, andthe recovered supernatant was centrifuged at 10,000 rpm at 4° C. for 1hour and then filtered using PES Filter Unit (manufactured by NALGENE)having a pore diameter of 0.22 μm with 150 ml capacity.

Prosep-A HighCapacity (manufactured by bioPROCESSING) was packed in a0.8 cm diameter column to a thickness of 2 cm and washed with 10 ml of0.1 mol/l citrate buffer (pH 3.0) and 10 ml of 1 mol/l glycine/NaOH-0.15mol/l NaCl buffer (pH 8.6) in that order to effect equilibration thecarrier. Next, 100 ml of each of the culture supernatant was passedthrough the column and washed with 50 ml of 1 mol/l glycine/NaOH-0.15mol/l NaCl buffer (pH 8.6). After washing them, the antibody absorbed toProsep-A was eluted using 2.5 ml of a 0.1 mol/l citrate buffer (pH 3.0),the eluate was fractionated at 500 μl and each fraction was neutralizedby mixing with 100 μl of 2 mol/l Tris-HCl (pH 8.5). Two fractionscontaining the antibody at a high concentration (1.2 ml in total) wereselected by the BCA method [Anal. Biochem., 150, 76 (1985)], combinedand then dialyzed against 10 mol/l citrate buffer (pH 6.0) at 4° C. fora whole day and night. After the dialysis, the antibody solution wasrecovered and subjected to sterile filtration using a 0.22 μm pore sizeMillex GV (manufactured by MILLIPORE).

(5) In Vitro Cytotoxic Activity (ADCC Activity) of Antibody Produced byMouse α-1,6-Fucosyltransferase (FUT8) Gene Over-Expressing Cell Line

In order to evaluate in vitro cytotoxic activity of the anti-GD3antibodies purified in the item (4), ADCC activity was measured using aGD3-positive cell, human melanoma cultured cell line G-361 (RCB0991,Cell Bank at The Institute of Physical and Chemical Research).

The G-361 cells subcultured in RPMI1640 medium (manufactured by LifeTechnologies) containing 10% fetal bovine serum (manufactured by LifeTechnologies) (hereinafter referred to as “RPMI1640-FBS(10)”) weresuspended in 500 μl of RPMI1640-FBS(10) at a density of 1×10⁶ cells, and3.7 MBq of Na₂ ⁵¹CrO₄ was added thereto, followed by culturing at 37° C.for 30 minutes for labeling the cells with a radioisotope. Aftercentrifugation at 1,200 rpm for 5 minutes, the supernatant was discardedand the target cells were suspended in 5 ml of RPMI1640-FBS(10). Thewashing step was repeated three times and then the cell suspension wasincubated for 30 minutes on ice for spontaneous dissociation of theradioactive substance. The washing step was again repeated twice andthen the cells were suspended in 5 ml of RPMI1640-FBS(10) to therebyprepare 2×10⁵ cells/ml of a target cell suspension.

On the other hand, 30 ml of peripheral blood was collected from ahealthy person and gently mixed with 0.5 ml of heparin sodium(manufactured by Shimizu Pharmaceutical) and then mixed with 30 ml ofphysiological saline (manufactured by Otsuka Pharmaceutical). After themixing, 10 ml of the mixture was gently overlaid on 4 ml of Lymphoprep(manufactured by NYCOMED PHARMA AS) and centrifuged at room temperatureat 2,000 rpm for 30 minutes. The separated mononuclear cell fractionswere collected from the centrifugation tubes, combined and thensuspended in 30 ml of RPMI1640-FBS(10). After centrifugation at roomtemperature at 1,200 rpm for 5 minutes, the supernatant was discardedand the cells were suspended in 20 ml of RPMI1640-FBS(10). The washingstep was repeated twice and then 2×10⁶ cells/ml of an effector cellsuspension was prepared using RPMI1640-FBS(10).

The target cell suspension was dispensed at 50 μl (1×10⁴ cells/well)into each well of a 96 well U-bottom plate (manufactured by Falcon).Subsequently, the effector cell suspension was dispensed at 100 μl(2×10⁵ cells/well) into each well to thereby adjust the ratio of theeffector cells to the target cells to 20:1. Next, using a 10 M citratebuffer (pH 6.0), a series of dilution solution of 0.01 μg/ml, 0.1 μg/ml,1 μg/ml and 10 μg/ml of each anti-GD3 antibody obtained in the item (4)was prepared, and the diluted solutions were dispensed at 50 μl into thewells to give final concentrations of 0.0025 μg/ml, 0.025 μg/ml, 0.25μg/ml and 2.5 μg/ml, respectively. After carrying out the reaction at37° C. for 4 hours in 5% CO₂, the plate was centrifuged at 1,200 rpm for5 minutes. Into a 12 mm diameter RIA tube (manufactured by IWAKI), 50 μlof the supernatant in each well was transferred and, and the amount ofthe dissociated ⁵¹Cr was measured using MINAX-7 auto-gamma counter 5550(manufactured by PACKARD).

Also, the amount of the spontaneously dissociated ⁵¹Cr was calculated bycarrying out the same reaction in a reaction mixture in which 150 μl ofRPMI1640-FBS(10) was added instead of the effector cell suspension andantibody solution. The amount of the total dissociated ⁵¹Cr wascalculated by carrying out the same reaction in a reaction mixture inwhich 100 μl of 1 N hydrochloric acid and 50 μl of RPMI1640-FBS(10) wereadded instead of the effector cell suspension and antibody solution.Using these values, the ADCC activity was calculated based on theformula (II) described in the item 2β) of Example 2.

FIG. 29 shows ADCC activity of each of the anti-GD3 antibodies for G-361cell. Three cell lines mfFUT8-1, mfFUT8-2 and mfFUT8-4 having a low FUT8expression level as shown in FIG. 28 showed potent ADCC activityequivalent to that of the negative control pAGE249-introduced cell line.On the other hand, other three cell lines mfFUT8-3, mfFUT8-6 andmfFUT8-7 having a high FUT8 expression level as shown in FIG. 28 showedlow ADCC activity equivalent to that of the anti-GD3 antibody producedfrom CHO cell. Based on these results, it was shown that the ADCCactivity of produced antibodies can be controlled by regulating theexpression level of FUT8 in host cells.

(6) Sugar Chain Analysis of Antibody Produced by Mouseα-1,6-Fucosyltransferase (FUT8) Gene Over-Expressing Cell Line

Sugar chains of the anti-GD3 antibodies purified in the item (4) wereanalyzed. The sugar chains binding to the antibodies produced bymfFUT8-6 and pAGE249-introduced cell lines were cleaved from proteins bysubjecting the antibodies to hydrazinolysis [Method of Enzymology, 83,263 (1982)]. After removing hydrazine by evaporation under a reducedpressure, N-acetylation was carried out by adding an aqueous ammoniumacetate solution and acetic anhydride. After freeze-drying, fluorescencelabeling by 2-aminopyridine [J. Biochem., 95, 197 (1984)] was carryingout. A fluorescence-labeled sugar chain group (PA-treated sugar chaingroup) was separated from excess reagents using Superdex Peptide HR10/30 column (manufactured by Pharmacia). The sugar chain fractions weredried using a centrifugation concentrator and used as a purifiedPA-treated sugar chain group. Next, the purified PA-treated sugar chaingroup was subjected to reverse phase HPLC analysis using a CLC-ODScolumn (manufactured by Shimadzu) (FIG. 30). When calculated from thepeak area, the content of α-1,6-fucose-free sugar chains in mfFUT8-6 was10%, and the content of α-1,6-fucose-bound sugar chains was 90%. Thecontent of α-1,6-fucose-free sugar chains in pAGE249 was 20%, and thecontent of α-1,6-fucose-bound sugar chains was 80%. Based on theseresults, it was found that the content of α-1,6-fucose-bound sugarchains of a produced antibody is increased by over-expressing the FUT8gene.

FIG. 30 shows elution patterns obtained by carrying out reverse phaseHPLC analysis of each of PA-treated sugar chains prepared fromantibodies produced by mfFUT8-6 and pAGE249-introduced cell lines. FIGS.30A and 30B show elution patterns of mfFUT8-6 and pAGE249, respectively.The relative fluorescence intensity and the elution time are plotted asthe ordinate and the abscissa, respectively. Using a sodium phosphatebuffer (pH 3.8) as buffer A and a sodium phosphate buffer (pH 3.8)+0.5%1-butanol as buffer B, the analysis was carried out by the followinggradient.

Time (minute) 0 80 90 90.1 120 Buffer B (%) 0 60 60 0 0

Peaks (i) to (ix) shown in FIG. 30 and FIG. 31 show the followingstructures.

GlcNAc, Gal, Man, Fuc and PA indicate N-acetylglucosamine, galactose,mannose, fucose and a pyridylamino group, respectively. In FIGS. 30 and31, the ratio of the α-1,6-fucose-free sugar chain group was calculatedfrom the area occupied by the peaks (i) to (iv) among (i) to (ix), andthe ratio of the α-1,6-fucose-bound sugar chain group from the areaoccupied by the peaks (v) to (ix) among (i) to (ix).

Example 12 Preparation of CHO Cell α-1,6-Fucosyltransferase (FUT8) Gene

(1) Preparation of CHO Cell α-1,6-Fucosyltransferase (FUT8) cDNASequence

From a single-stranded cDNA prepared from CHO/DG44 cells on the 2nd dayof culturing in Example 9(1), Chinese hamster FUT8 cDNA was obtained bythe following procedure (FIG. 32).

First, a forward primer specific for a 5′-terminal non-translationregion (shown in SEQ ID NO:21) and a reverse primer specific for a3′-terminal non-translation region (shown in SEQ ID NO:22) were designedfrom a mouse FUT8 cDNA sequence (GenBank, AB025198).

Next, 25 μl of a reaction solution [ExTaq buffer (manufactured by TakaraShuzo), 0.2 mmol/l dNTPs, 4% DMSO and 0.5 μmol/l specific primers (SEQID NOs:21 and 22)] containing 1 μl of the CHO/DG44 cell-derived cDNA wasprepared and PCR was carried out using a DNA polymerase ExTaq(manufactured by Takara Shuzo). The PCR was carried out by heating at94° C. for 1 minute, subsequent 30 cycles of heating at 94° C. for 30seconds, 55° C. for 30 seconds and 72° C. for 2 minutes as one cycle,and final heating at 72° C. for 10 minutes.

After the PCR, the reaction solution was subjected to 0.8% agarose gelelectrophoresis, and a specific amplified fragment of about 2 Kb waspurified. Into a plasmid pCR2.1, 4 μl of the DNA fragment was employedto insert in accordance with the instructions attached to TOPO TACloning Kit (manufactured by Invitrogen), and E. coli DH5α wastransformed with the reaction solution. Plasmid DNAs were isolated inaccordance with a known method from cDNA-inserted 8 clones among theobtained kanamycin-resistant colonies.

The nucleotide sequence of each cDNA inserted into the plasmid wasdetermined using DNA Sequencer 377 (manufactured by Parkin Elmer) andBigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufacturedby Parkin Elmer) in accordance with the method of the manufacture'sinstructions. It was confirmed by the method that all of the insertedcDNAs encode a sequence containing the full ORF of CHO cell FUT8. Amongthese, a plasmid DNA containing absolutely no reading error of bases bythe PCR in the sequences was selected. Herein, the plasmid is referredto as CHfFUT8-pCR2.1. The determined nucleotide sequence and the aminoacid sequence of the cDNA of CHO FUT8 are shown in SEQ ID NOs:1 and 23,respectively.

(2) Preparation of CHO Cell α-1,6-Fucosyltransferase (FUT8) GenomicSequence

Using the ORF full length cDNA fragment of CHO cell FUT8 obtained in theitem (1) as a probe, a CHO cell FUT8 genomic clone was obtained inaccordance with a known genome screening method described, e.g., inMolecular Cloning, Second Edition, Current Protocols in MolecularBiology, A Laboratory Manual, Second Edition (1989). Next, afterdigesting the obtained genomic clone using various restriction enzymes,the Southern hybridization was carried out using an AfaI-Sau3AI fragment(about 280 bp) containing initiation codon of the CHO cell FUT8 cDNA asa probe, and then a XbaI-XbaI fragment (about 2.5 Kb) and a SacI-SacIfragment (about 6.5 Kb) were selected from restriction enzyme fragmentsshowing positive reaction, inserted into pBluescript II KS(+)(manufactured by Stratagene), respectively.

The nucleotide sequence of each of the obtained genomic fragments wasdetermined using DNA Sequencer 377 (manufactured by Parkin Elmer) andBigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufacturedby Parkin Elmer) in accordance with the method of the manufacture'sinstructions. Thereby, it was confirmed that the XbaI-XbaI fragmentencodes a sequence of an upstream intron of about 2.5 Kb containing exon2 of the CHO cell FUT8, and the SacI-SacI fragment encodes a sequence ofa downstream intron of about 6.5 Kb containing exon 2 of the CHO cellFUT8. Herein, the plasmid containing XbaI-XbaI fragment is referred toas pFUT8fgE2-2, and the plasmid containing SacI-SacI fragment isreferred to as pFUT8fgE2-4. The determined nucleotide sequence (about9.0 Kb) of the genome region containing exon 2 of the CHO cell FUT8 isshown in SEQ ID NO:3.

Example 13 Preparation of CHO Cell in which α-1,6-Fucose TransferaseGene is Disrupted and Production of Antibody Using the Cell

A CHO cell from which the genome region comprising the CHO cellα-1,6-fucosyltransferase (FUT8) gene exon 2 was deleted was prepared andthe ADCC activity of an antibody produced by the cell was evaluated.

1. Construction of Chinese Hamster α-1,6-Fucosyltransferase (FUT8) GeneExon 2 Targeting Vector Plasmid pKOFUT8Puro(1) Construction of Plasmid ploxPPuro

A plasmid ploxPPuro was constructed by the following procedure (FIG.33).

In 35 μl of NEBuffer 4 (manufactured by New England Biolabs), 1.0 μg ofa plasmid pKOSelectPuro (manufactured by Lexicon) was dissolved, and 20units of a restriction enzyme AscI (manufactured by New England Biolabs)were added thereto, followed by digestion reaction at 37° C. for 2hours. After the digestion reaction, the solution was subjected to 0.8%(w/v) agarose gel electrophoresis to purify a DNA fragment of about 1.5Kb containing a puromycin resistance gene expression unit.

On the other hand, 1.0 μg of a plasmid ploxP described in JapanesePublished Examined Patent Application No. 314512/99 was dissolved in 35μl of NEBuffer 4 (manufactured by New England Biolabs), and 20 units ofa restriction enzyme AscI (manufactured by New England Biolabs) wereadded thereto, followed by digestion reaction at 37° C. for 2 hours.After the digestion reaction, the solution was subjected to 0.8% (w/v)agarose gel electrophoresis to purify a DNA fragment of about 2.0 Kb.

The obtained AscI-AscI fragment (4.5 μl, about 1.5 Kb) derived from theplasmid pKOSelectPuro, 0.5 μl of the AscI-AscI fragment (about 2.0 Kb)derived from the plasmid ploxP and 5.0 μl of Ligation High (manufacturedby Toyobo) were mixed, followed by ligation reaction at 16° C. for 30minutes. E. coli DH5α was transformed using the reaction solution, and aplasmid DNA was isolated in accordance with a known method from theobtained ampicillin-resistant clones. Herein, the plasmid is referred toas ploxPPuro.

(2) Construction of Plasmid pKOFUT8gE2-1

A plasmid pKOFUT8gE2-1 was constructed by the following procedure (FIG.34), using the plasmid pFUT8fgE2-obtained in Example 12(2) having agenome region comprising exon 2 of Chinese hamster FUT8.

In 35 μl of NEBuffer 1 (manufactured by New England Biolabs) containing100 μg/ml of BSA (manufactured by New England Biolabs), 2.0 μg of theplasmid pFUT8fgE2-2 was dissolved, and 20 units of a restriction enzymeSacI (manufactured by New England Biolabs) were added thereto, followedby digestion reaction at 37° C. for 2 hours. A DNA fragment wasrecovered from the reaction solution by ethanol precipitation anddissolved in 35 μl of NEBuffer 2 (manufactured by New England Biolabs)containing 100 μg/ml of BSA (manufactured by New England Biolabs), and20 units of a restriction enzyme EcoRV (manufactured by New EnglandBiolabs) were added thereto, followed by digestion reaction at 37° C.for 2 hours. After the digestion reaction, the solution was subjected to0.8% (w/v) agarose gel electrophoresis to purify a DNA fragment of about1.5 Kb.

Separately, 1.0 μg of a plasmid LITMUS28 (manufactured by New EnglandBiolabs) was dissolved in 35 μl of NEBuffer 1 (manufactured by NewEngland Biolabs) containing 100 μg/ml of BSA (manufactured by NewEngland Biolabs), and 20 units of a restriction enzyme SacI(manufactured by New England Biolabs) were added thereto, followed bydigestion reaction at 37° C. for 2 hours. A DNA fragment was recoveredfrom the reaction solution by ethanol precipitation and dissolved in 35μl of NEBuffer 2 (manufactured by New England Biolabs) containing 100μg/ml of BSA (manufactured by New England Biolabs), and 20 units of arestriction enzyme EcoRV (manufactured by New England Biolabs) wereadded thereto, followed by digestion reaction at 37° C. for 2 hours.After the digestion reaction, the solution was subjected to 0.8% (w/v)agarose gel electrophoresis to purify a DNA fragment of about 2.8 Kb.

The obtained EcoRV-SacI fragment (4.5 μl, about 1.5 Kb) derived from theplasmid pFUT8fgE2-2, 0.5 μl of the EcoRV-SacI fragment (about 2.8 Kb)derived from the plasmid LITMUS28 and 5.0 μl of Ligation High(manufactured by Toyobo) were mixed, followed by ligation reaction at16° C. for 30 minutes. E. coli DH5α was transformed using the reactionsolution, and a plasmid DNA was isolated in accordance with a knownmethod from the obtained ampicillin-resistant clones. Herein, theplasmid is referred to as pKOFUT8gE2-1.

(3) Construction of Plasmid pKOFUT8gE2-2

A plasmid pKOFUT8gE2-2 was constructed by the following procedure (FIG.35), using the plasmid pKOFUT8gE2-1 obtained in the item (2).

In 30 μl of NEBuffer 2 (manufactured by New England Biolabs) containing100 μg/ml of BSA (manufactured by New England Biolabs), 2.0 μg of theplasmid pKOFUT8gE2-1 was dissolved, and 20 units of a restriction enzymeEcoRV (manufactured by New England Biolabs) were added thereto, followedby digestion reaction at 37° C. for 2 hours. A DNA fragment wasrecovered from the reaction solution by ethanol precipitation anddissolved in 30 μl of NEBuffer 1 (manufactured by New England Biolabs)containing 100 μg/ml of BSA (manufactured by New England Biolabs), and20 units of a restriction enzyme KpnI (manufactured by New EnglandBiolabs) were added thereto, followed by digestion reaction at 37° C.for 2 hours. After the digestion reaction, the solution was subjected to0.8% (w/v) agarose gel electrophoresis to purify a DNA fragment of about1.5 Kb.

Separately, 1.0 μg of the plasmid ploxPPuro was dissolved in 30 μl ofNEBuffer 4 (manufactured by New England Biolabs), and 20 units of arestriction enzyme HpaI (manufactured by New England Biolabs) were addedthereto, followed by digestion reaction at 37° C. for 2 hours. A DNAfragment was recovered from the reaction solution by ethanolprecipitation and dissolved in 30 μl of NEBuffer 1 (manufactured by NewEngland Biolabs) containing 100 μg/ml of BSA (manufactured by NewEngland Biolabs), and 20 units of a restriction enzyme KpnI(manufactured by New England Biolabs) were added thereto, followed bydigestion reaction at 37° C. for 2 hours. After the digestion reaction,the solution was subjected to 0.8% (w/v) agarose gel electrophoresis topurify a DNA fragment of about 3.5 Kb.

A 4.0 μl portion of the obtained EcoRV-KpnI fragment (about 1.5 Kb)derived from the plasmid pKOFUT8gE2-1, 1.0 μl of the HpaI-KpnI fragment(about 3.5 Kb) derived from the plasmid ploxPPuro and 5.0 μl of LigationHigh (manufactured by Toyobo) were mixed and allowed to undergo theligation reaction at 16° C. for 30 minutes. E. coli DH5α a wastransformed using the reaction solution, and a plasmid DNA was isolatedin accordance with a known method from the obtained ampicillin-resistantclones. Herein, the plasmid is referred to pKOFUT8gE2-2.

(4) Construction of Plasmid pscFUT8gE2-3

A plasmid pscFUT8gE2-3 was constructed by the following procedure (FIG.36), using the plasmid pFUT8fgE2-obtained in Example 12(2) having agenome region comprising exon 2 of Chinese hamster FUT8.

In 35 μl of NEBuffer 1 (manufactured by New England Biolabs), 2.0 μg ofthe plasmid pFUT8fgE2-4 was dissolved, and 20 units of a restrictionenzyme HpaII (manufactured by New England Biolabs) were added thereto,followed by digestion reaction at 37° C. for 2 hours. A DNA fragment wasrecovered from the reaction solution by ethanol precipitation, and thenthe DNA termini were changed to blunt ends using Blunting High(manufactured by Toyobo) in accordance with the manufacture'sinstructions. The DNA fragment was recovered by carrying outphenol/chloroform extraction and ethanol precipitation and dissolved in35 μl of NEBuffer 2 (manufactured by New England Biolabs), and 20 unitsof a restriction enzyme HindIII (manufactured by New England Biolabs)were added thereto, followed by digestion reaction at 37° C. for 2hours. After the digestion reaction, the solution was subjected to 0.8%(w/v) agarose gel electrophoresis to purify a DNA fragment of about 3.5Kb.

On the other hand, 1.0 μg of a plasmid LITMUS39 (manufactured by NewEngland Biolabs) was dissolved in 35 μl of NEBuffer 2 (manufactured byNew England Biolabs), and the solution was mixed with 20 units of arestriction enzyme EcoRV (manufactured by New England Biolabs) and 20units of a restriction enzyme HindIII (manufactured by New EnglandBiolabs) and subjected to the digestion reaction at 37° C. for 2 hours.After the digestion reaction, the solution was subjected to 0.8% (w/v)agarose gel electrophoresis to purify a DNA fragment of about 2.8 Kb.

The obtained HpaII-HindIII fragment (4.0 μl, about 3.5 Kb) derived fromthe plasmid pFUT8fgE2-4, 1.0 μl of the EcoRV-HindIII fragment (about 2.8Kb) derived from the plasmid LITMUS39 and 5.0 μl of Ligation High(manufactured by Toyobo) were mixed, followed by ligation reaction at16° C. for 30 minutes. E. coli DH5α was transformed using the reactionsolution, and a plasmid DNA was isolated in accordance with a knownmethod from the obtained ampicillin-resistant clones. Herein, theplasmid is referred to pscFUT8gE2-3.

(5) Construction of Plasmid pKOFUT8gE2-3

A plasmid pKOFUT8gE2-3 was constructed by the following procedure (FIG.37), using the plasmid pFUT8fgE2-obtained in Example 12(2) having agenome region comprising exon 2 of Chinese hamster FUT8.

In 35 μl of NEBuffer for EcoRI (manufactured by New England Biolabs),2.0 μg of the plasmid pFUT8fgE2-4 was dissolved, and 20 units of arestriction enzyme EcoRI (manufactured by New England Biolabs) and 20units of a restriction enzyme HindIII (manufactured by New EnglandBiolabs) were added thereto, followed by digestion reaction at 37° C.for 2 hours. After the digestion reaction, the solution was subjected to0.8% (w/v) agarose gel electrophoresis to purify a DNA fragment of about1.8 Kb.

Separately, 1.0 μg of a plasmid pBluescript II KS(+) (manufactured byStratagene) was dissolved in 35 μl of NEBuffer for EcoRI (manufacturedby New England Biolabs), and 20 units of a restriction enzyme EcoRI(manufactured by New England Biolabs) and 20 units of a restrictionenzyme HindIII (manufactured by New England Biolabs) were added thereto,followed by digestion reaction at 37° C. for 2 hours. After thedigestion reaction, the solution was subjected to 0.8% (w/v) agarose gelelectrophoresis to purify a DNA fragment of about 3.0 Kb.

The obtained HindIII-EcoRI fragment (4.0 μl, about 1.8 Kb) derived fromthe plasmid pFUT8fgE2-4, 1.0 μl of the HindIII-EcoRI fragment (about 3.0Kb) derived from the plasmid pBluescript II KS(+) and 5.0 μl of LigationHigh (manufactured by Toyobo) were mixed, followed by ligation reactionat 16° C. for 30 minutes. E. coli DH5α was transformed using thereaction solution, and a plasmid DNA was isolated in accordance with aknown method from the obtained ampicillin-resistant clones. Herein, theplasmid is referred to pKOFUT8gE2-3.

(6) Construction of Plasmid pKOFUT8gE2-4

A plasmid pKOFUT8gE2-4 was constructed by the following procedure (FIG.38), using the plasmids pscFUT8fgE2-3 and pKOFUT8gE2-3 obtained in theitems (4) and (5).

In 35 μl of NEBuffer for SalI (manufactured by New England Biolabs)containing 100 μg/ml of BSA (manufactured by New England Biolabs), 1.0μg of the plasmid pscFUT8gE2-3 was dissolved, and 20 units of arestriction enzyme SalI (manufactured by New England Biolabs) were addedthereto, followed by digestion reaction at 37° C. for 2 hours. A DNAfragment was recovered from the reaction solution by ethanolprecipitation and dissolved in 30 μl of NEBuffer 2 (manufactured by NewEngland Biolabs) containing 100 μg/ml of BSA (manufactured by NewEngland Biolabs), and 20 units of a restriction enzyme HindIII(manufactured by New England Biolabs) were added thereto, followed bydigestion reaction at 37° C. for 2 hours. After the digestion reaction,the solution was subjected to 0.8% (w/v) agarose gel electrophoresis topurify a DNA fragment of about 3.6 Kb.

Separately, 1.0 μg of the plasmid pKOFUT8gE2-3 was dissolved in 35 μl ofNEBuffer for SalI (manufactured by New England Biolabs), and 20 units ofa restriction enzyme SalI (manufactured by New England Biolabs) wereadded thereto, followed by digestion reaction at 37° C. for 2 hours. ADNA fragment was recovered from the reaction solution by ethanolprecipitation and dissolved in 35 μl of NEBuffer 2 (manufactured by NewEngland Biolabs), and 20 units of a restriction enzyme HindIII(manufactured by New England Biolabs) were added thereto, followed bydigestion reaction at 37° C. for 2 hours. After the digestion reaction,35 μl of 1 mol/l Tris-HCl buffer (pH 8.0) and 3.5 μl of E. coliC15-derived alkaline phosphatase (manufactured by Takara Shuzo) wereadded thereto, followed by reaction at 65° C. for 30 minutes todephosphorylate the DNA termini. After the dephosphorylation treatment,a DNA fragment was recovered by carrying out phenol/chloroformextraction and ethanol precipitation and dissolved in 10 μl of sterilewater.

The obtained SalI-HindIII fragment (4.0 μl, about 3.1 Kb) derived fromthe plasmid pscFUT8gE2-3, 1.0 μl of the SalI-HindIII fragment (about 4.8Kb) derived from the plasmid pKOFUT8gE2-3 and 5.0 μl of Ligation High(manufactured by Toyobo) were mixed, followed by ligation reaction at16° C. for 30 minutes. E. coli DH5α was transformed using the reactionsolution, and a plasmid DNA was isolated in accordance with a knownmethod from the obtained ampicillin-resistant clones. Herein, theplasmid is referred to pKOFUT8gE2-4.

(7) Construction of Plasmid pKOFUT8gE2-5

A plasmid pKOFUT8gE2-5 was constructed by the following procedure (FIG.39), using the plasmids pKOFUT8gE2-2 and pKOFUT8gE2-4 obtained in theitems (3) and (6).

In 30 μl of NEBuffer 4 (manufactured by New England Biolabs), 1.0 μg ofthe plasmid pKOFUT8gE2-2 was dissolved, and 20 units of a restrictionenzyme SmaI (manufactured by New England Biolabs) were added thereto,followed by digestion reaction at 25° C. for 2 hours. A DNA fragment wasrecovered from the reaction solution by ethanol precipitation anddissolved in 30 μl of NEBuffer 2 (manufactured by New England Biolabs),and 20 units of a restriction enzyme BamHI (manufactured by New EnglandBiolabs) were added thereto, followed by digestion reaction at 37° C.for 2 hours. After the digestion reaction, 30 μl of 1 mol/l Tris-HClbuffer (pH 8.0) and 3.0 μl of E. coli C15-derived alkaline phosphatase(manufactured by Takara Shuzo) were added thereto, followed by reactionat 65° C. for hour to dephosphorylate the DNA termini. After thedephosphorylation treatment, the DNA fragment was recovered by carryingout phenol/chloroform extraction and ethanol precipitation and dissolvedin 10 μl of sterile water.

Separately, 1.0 μg of the plasmid pKOFUT8gE2-4 was dissolved in 30 μl ofNEBuffer 4 (manufactured by New England Biolabs), and 20 units of arestriction enzyme SmaI (manufactured by New England Biolabs) were addedthereto, followed by digestion reaction at 25° C. for 2 hours. A DNAfragment was recovered from the reaction solution by ethanolprecipitation and dissolved in 30 μl of NEBuffer 2 (manufactured by NewEngland Biolabs), and 20 units of a restriction enzyme BamHI(manufactured by New England Biolabs) were added thereto, followed bydigestion reaction at 37° C. for 2 hours. After the digestion reaction,the solution was subjected to 0.8% (w/v) agarose gel electrophoresis topurify a DNA fragment of about 5.2 Kb.

The obtained SmaI-BamHI fragment (0.5 μl, about 5.0 Kb) derived from theplasmid pKOFUT8gE2-2, 4.5 μl of the SmaI-BamHI fragment (about 5.4 Kb)derived from the plasmid pKOFUT8gE2-4 and 5.0 μl of Ligation High(manufactured by Toyobo) were mixed, followed by ligation reaction at16° C. for 15 hours. E. coli DH5α was transformed using the reactionsolution, and a plasmid DNA was isolated in accordance with a knownmethod from the obtained ampicillin-resistant clones. Herein, theplasmid is referred to pKOFUT8gE2-5.

(8) Construction of Plasmid pKOFUT8Puro

A plasmid pKOFUT8Puro was constructed by the following procedure (FIG.40), using the plasmid pKOFUT8gE2-5 obtained in the item (7).

In 50 μl of NEBuffer 4 (manufactured by New England Biolabs), 1.0 μg ofa plasmid pKOSelectDT (manufactured by Lexicon) was dissolved, and 16units of a restriction enzyme RsrII (manufactured by New EnglandBiolabs) were added thereto, followed by digestion reaction at 37° C.for 2 hours. After the digestion reaction, the solution was subjected to0.8% (w/v) agarose gel electrophoresis to purify a DNA fragment of about1.2 Kb comprising a diphtheria toxin expression unit.

Separately, 1.0 μg of the plasmid pKOFUT8gE2-5 was dissolved in 50 μl ofNEBuffer 4 (manufactured by New England Biolabs), and 16 units of arestriction enzyme RsrII (manufactured by New England Biolabs) wereadded thereto, followed by digestion reaction at 37° C. for 2 hours.After the digestion reaction, 30 μl of 1 mol/l Tris-HCl buffer (pH 8.0)and 3.0 μl of E. coli C15-derived alkaline phosphatase (manufactured byTakara Shuzo) were added thereto, followed by reaction at 65° C. for 1hour to dephosphorylate the DNA termini. After the dephosphorylationtreatment, the DNA fragment was recovered by carrying outphenol/chloroform extraction and ethanol precipitation and dissolved in10 μl of sterile water.

The obtained RsrII-RsrII fragment (1.0 μl, about 1.2 Kb) derived fromthe plasmid pKOSelectDT, 1.0 μl of the RsrII-RsrII fragment (about 10.4Kb) derived from the plasmid pKOFUT8gE2-5, 3.0 μl of sterile water and5.0 μl of Ligation High (manufactured by Toyobo) were mixed, followed byligation reaction at 16° C. for 30 minutes. E. coli DH5α was transformedusing the reaction solution, and a plasmid DNA was isolated inaccordance with a known method from the obtained ampicillin-resistantclones. Herein, the plasmid is referred to pKOFUT8Puro.

2. Preparation of CHO Cell in which One Copy of the Genome RegionContaining α-1,6-Fucosyltransferase (FUT8) Gene Exon 2 Was Disrupted

(1) Introduction of Targeting Vector

A Chinese hamster FUT8 genome region targeting vector pKOFUT8Puroconstructed in the item 1 of this Example was introduced into the strain5-03 prepared in the item 1(2) of Example 8.

A gene of the plasmid pKOFUT8Puro was introduced into the strain 5-03 asdescribed below in accordance with the electroporation method[Cytotechnology, 3, 133 (1990)]. First, 150 μg of the plasmidpKOFUT8Puro was dissolved in 1.8 ml of NEBuffer for SalI (manufacturedby New England Biolabs), and 600 units of a restriction enzyme SalI(manufactured by New England Biolabs) were added thereto, followed bydigestion reaction at 37° C. for 5 hours to obtain a linear fragment.The reaction solution was extracted with phenol/chloroform extraction,followed by ethanol precipitation, and the recovered linear plasmid wasmade into a 1 μg/μl aqueous solution. Separately, the strain 5-03 wassuspended in a K-PBS buffer (137 mmol/l KCl, 2.7 mmol/l NaCl, 8.1 mmol/lNa₂HPO₄, 1.5 mmol/l KH₂PO₄, 4.0 mmol/l MgCl₂) to give a density of 8×10⁷cells/ml. After mixing 200 μl of the cell suspension (1.6×10⁶ cells)with 4 μl (4 μg) of the linear plasmid, an entire volume of the cell-DNAmixture was transferred into Gene Pulser Cuvette (inter-electrodedistance, 2 mm) (manufactured by BIO-RAD) and then the electroporationwas carried out using a cell fusion apparatus Gene Pulser (manufacturedby BIO-RAD) at 350 V pulse voltage and 250 μF electric capacity. Aftercarrying out the electroporation using 30 cuvettes in the same manner,the cell suspension was suspended in IMDM medium (manufactured by LifeTechnologies) supplemented with 10% fetal bovine serum (manufactured byLife Technologies) and 1× concentration HT supplement (manufactured byLife Technologies) and inoculated into 30 adhesion cell culture dishesof 10 cm in diameter (manufactured by Falcon). After culturing them at37° C. for 24 hours in 5% CO₂, the culture supernatant was removed, andIMDM medium (manufactured by Life Technologies) supplemented with 15μg/ml puromycin (manufactured by SIGMA) and 10% fetal bovine dialyzedserum (manufactured by Life Technologies) was dispensed in 10 mlportions. After culturing them for 10 days while repeating the mediumexchange at intervals of 3 to 4 days, puromycin-resistant cell lineswere obtained.

(2) Preparation of Targeting Vector-Introduced Cell Lines

Arbitrary 900 colonies were obtained as follows from thepuromycin-resistant cell lines obtained in the item (1).

First, culture supernatant was removed from the 10 cm dish in whichcolony of puromycin-resistant cell lines were formed and 7 ml of aphosphate buffer was added to the dish which was subsequently set undera stereoscopic microscope. Next, each colony was scratched and sucked upusing Pipetteman (manufactured by GILSON) and transferred into a 96 wellround-bottom plate (manufactured by Falcon). After a trypsin-treatment,each clone was inoculated into a well flat-bottom plate for adhesioncell culture use (manufactured by Iwaki Glass) and cultured for 1 weekusing IMDM medium (manufactured by Life Technologies) supplemented with15 μg/ml puromycin (manufactured by SIGMA) and 10% fetal bovine dialyzedserum (manufactured by Life Technologies).

After culturing them, each clone in the plate was subjected to trypsintreatment and then mixed with two volumes of a freeze drying medium (20%DMSO, 40% fetal bovine serum, 40% IMDM). A half of the mixture wasinoculated into a 96 well flat-bottom plate for adhesion cell culture(manufactured by Iwaki Glass) as a replica plate, while the remaininghalf of the mixture was subjected to cryopreservation as master plates.The replica plate was cultured for 1 week using IMDM medium(manufactured by Life Technologies) supplemented with 15 μg/ml ofpuromycin (manufactured by SIGMA) and 10% fetal bovine dialyzed serum(manufactured by Life Technologies).

(3) Diagnosis of Homologous Recombination by Genomic PCR

Diagnosis of homologous recombination in the 900 clones obtained in theitem (2) was carried out by genomic PCR.

First, genome DNA of each clone was prepared from the replica plateprepared in the item (2) in accordance with a known method [AnalyticalBiochemistry, 201, 331 (1992)] and dissolved overnight in 30 μl of aTE-RNase buffer (pH 8.0) (10 mmol/l Tris-HCl, 1 mmol/l EDTA, 200 μg/mlRNase A). Also, a primer (shown in SEQ ID NO:26) which binds to asequence outside the targeting vector homologous region among the FUT8genome region obtained in Example 12 and a primer (shown in SEQ IDNO:27) which binds to the loxP sequence in the vector were designed.

Using a DNA polymerase ExTaq (manufactured by Takara Shuzo), 25 μl of areaction solution [ExTaq buffer (manufactured by Takara Shuzo), 0.2mmol/l dNTPs and 0.5 μmol/l gene-specific primers (SEQ ID NO:26 and SEQID NO:27)] containing 10 μl of each the above-prepared genome DNAsolution were prepared, and polymerase chain reaction (PCR) was carriedout. The PCR was carried out by heating at 94° C. for 3 minutes andsubsequent 38 cycles of heating using the reaction at 94° C. for 1minute, 60° C. for 1 minute and 72° C. for 2 minutes as one cycle.

After the PCR, the reaction solution was subjected to 0.8% (w/v) agarosegel electrophoresis, and a specifically amplifying fragment of about 1.7Kb containing a border region between the CHO cell genome region and thetargeting vector homologous region was identified as a positive clone.One positive clone was found by the method.

(4) Diagnosis of Homologous Recombination by Genome Southern Blotting

Diagnosis of homologous recombination in the 1 clone, whose positivesignal was confirmed in the item (3), was carried out by genome Southernblotting.

Among the master plates cryo-preserved in the item (2), a 96 well platecontaining the positive clone found in the item (3) was selected andincubated at 37° C. for 10 minutes in 5% CO₂. After the incubation,cells were collected from a well corresponding to the positive clone andinoculated into a 24 well flat bottom plate for adhesion cell(manufactured by Greiner). After culturing them for 1 week using IMDMmedium (manufactured by Life Technologies) supplemented with 15 μg/ml ofpuromycin (manufactured by SIGMA) and 10% fetal bovine dialyzed serum(manufactured by Life Technologies), the cells were inoculated into a 6well flat bottom plate for adhesion cell (manufactured by Greiner).Genome DNA was prepared from the clone in the plate in accordance with aknown method [Nucleic Acids Research, 3, 2303 (1976)] and dissolvedovernight in 150 μl of a TE-RNase buffer (pH 8.0) (10 mmol/l Tris-HCl, 1mmol/l EDTA, 200 μg/ml RNase A).

In 120 μl of NEBuffer 3 (manufactured by New England Biolabs), 12 μg ofthe obtained genome DNA was dissolved, and 25 unites of a restrictionenzyme PstI (manufactured by New England Biolabs) were added thereto,followed by digestion reaction at 37° C. overnight. A DNA fragment wasrecovered from the reaction solution by ethanol precipitation, dissolvedin 20 μl of TE buffer (pH 8.0) (10 mmol/l Tris-HCl, 1 mmol/l EDTA) andthen subjected to 0.8% (w/v) agarose gel electrophoresis. After theelectrophoresis, the genome DNA was transferred onto a nylon membrane inaccordance with a known method [Proc. Natl. Acad. Sci. USA, 76, 3683(1979)]. After completion of the transfer, the nylon membrane was heatedat 80° C. for 2 hours.

Separately, a probe used in the Southern blotting was prepared asfollows. First, primers (SEQ ID NOs:28 and 29) which bind to a sequenceoutside the targeting vector homologous region with the FUT8 genomeregion obtained in Example 12 were designed. Next, using a DNApolymerase ExTaq (manufactured by Takara Shuzo), 20 μl of a reactionsolution [ExTaq buffer (manufactured by Takara Shuzo), 0.2 mmol/l dNTPsand 0.5 μmol/l gene-specific primers (SEQ ID NOs:28 and 29)] containing4.0 ng of the plasmid pFUT8fgE2-obtained in Example 12(2) was prepared,and polymerase chain reaction (PCR) was carried out. The PCR was carriedout by heating at 94° C. for 1 minute and subsequent 25 cycles ofheating at 94° C. for 30 seconds, 55° C. for 30 seconds and 74° C. for 1minute as one cycle. After the PCR, the reaction solution was subjectedto 1.75% (w/v) agarose gel electrophoresis to purify a probe DNAfragment of about 230 bp. The obtained probe DNA solution (5 μl) waslabeled with a radioisotope using 1.75 MBq of [α-³²P]dCTP and MegaprimeDNA Labeling System, dCTP (manufactured by Amersham Pharmacia Biotech).

The hybridization was carried out as follows. First, the nylon membranewas sealed in a roller bottle, and pre-hybridization was carried out at65° C. for 3 hours by adding 15 ml of a hybridization solution [5×SSPE,50×Denhaldt's solution, 0.5% (w/v) SDS, 100 μg/ml salmon sperm DNA].Next, the ³²P-labeled probe DNA was heat-denatured and put into thebottle. Then, the nylon membrane was heated at 65° C. overnight.

After the hybridization, the nylon membrane was soaked in 50 ml of2×SSC-0.1% (w/v) SDS and heated at 65° C. for 15 minutes. Afterrepeating the washing step twice, the membrane was soaked in 50 ml of0.2×SSC-0.1% (w/v) SDS and heated at 65° C. for 15 minutes. After thewashing, the nylon membrane was exposed to an X-ray film at −80° C. fortwo nights for development.

By the restriction enzyme PstI treatment, a DNA fragment of about 4.4 Kbis formed from a wild type FUT8 allele. On the other hand, a DNAfragment of about 6.0 Kb is formed from an allele in which homologousrecombination with a targeting vector was generated.

By the method, such specific fragments of about 4.4 Kb and about 6.0 Kbwere found from the positive clone genome DNA in the item (3). Since thequantitative ratio of both fragments was 1:1, it was confirmed that theclone is a clone in which 1 copy of the FUT8 allele was disrupted.Hereinafter, the clone is referred to as the strain 1st.ΔFUT8 2-46.

3. Deletion of Drug Resistance Gene from CHO Cell in which Copy of theα-1,6-Fucosyltransferase (FUT8) Gene was Disrupted

(1) Introduction of Cre Recombinase Expression Vector

A Cre recombinase expression vector pBS185 (manufactured by LifeTechnologies) was introduced into the strain 1st.ΔFUT8 2-46 prepared inthe item 2 of this Example.

A gene of the plasmid pBS185 was introduced into the strain 1st.ΔFUT82-46 as follows in accordance with the electroporation method[Cytotechnology, 3, 133 (1990)]. First, the strain 1st.ΔFUT8 2-46 wassuspended in a K-PBS buffer (137 mmol/l KCl, 2.7 mmol/l NaCl, 8.1 mmol/lNa₂HPO₄, 1.5 mmol/l KH₂PO₄, 4.0 mmol/l MgCl₂) to give a density of 8×10⁷cells/ml. After mixing 200 μl of the cell suspension (1.6×10⁶ cells)with 4 μg of the plasmid pBS185, an entire volume of the cell-DNAmixture was transferred into Gene Pulser Cuvette (inter-electrodedistance, 2 mm) (manufactured by BIO-RAD) and then the gene transfer wascarried out using a cell fusion apparatus Gene Pulser (manufactured byBIO-RAD) at 350 V pulse voltage and 250 μF electric capacity. After thegene transfer, the cell suspension was suspended in 10 ml of IMDM medium(manufactured by Life Technologies) supplemented with 10% fetal bovineserum (manufactured by Life Technologies) and 1× concentration HTsupplement (manufactured by Life Technologies) and further diluted20,000 folds using the same medium. The cells were inoculated into 7adhesion cell culture dishes of 10 cm in diameter (manufactured byFalcon) and then cultured at 37° C. for 24 hours in 5% CO₂. Afterculturing them, the culture supernatant was removed and IMDM medium(manufactured by Life Technologies) supplemented with 10% fetal bovinedialyzed serum (manufactured by Life Technologies) was dispensed at 10ml. Culturing was carried out for 10 days while repeating the mediumexchange at intervals of 3 to 4 days.

(2) Preparation of Cre Recombinase Expression Vector-Introduced CellLines

Arbitrary 400 colonies were obtained as follows from the cell lineobtained in the item (1).

First, culture supernatant was removed from the 10 cm dish and 7 ml of aphosphate buffer was added to the dish which was subsequently set undera stereoscopic microscope. Next, each colony was scratched and sucked upusing Pipetteman (manufactured by GILSON) and transferred into a 96 wellround-bottom plate (manufactured by Falcon). After a trypsin-treatment,each clone was inoculated into a well flat-bottom plate for adhesioncell culture (manufactured by Iwaki Glass) and cultured for 1 week usingIMDM medium (manufactured by Life Technologies) supplemented with 10%fetal bovine dialyzed serum (manufactured by Life Technologies).

After the culturing, each clone in the plate was subjected to trypsintreatment and then mixed with two volumes of a freeze drying medium (20%DMSO, 40% fetal bovine serum, 40% IMDM). A half of the mixture wasinoculated into a 96 well flat-bottom plate for adhesion cell cultureuse (manufactured by Iwaki Glass) to prepare a replica plate, while theremaining half was subjected to cryopreservation as a master plate.

Next, the replica plate was cultured for 6 days using IMDM medium(manufactured by Life Technologies) supplemented with 15 μg/ml ofpuromycin (manufactured by SIGMA) and 10% fetal bovine dialyzed serum(manufactured by Life Technologies). A positive clone from which thepuromycin resistance gene interposed between loxP sequences waseliminated by the expression of Cre recombinase dies out in the presenceof puromycin. By the selection method, 91 positive clones were found.

(3) Diagnosis of Drug Resistance Gene Elimination by Genome SouthernBlotting

Diagnosis of drug resistance gene elimination by the genome Southernblotting was carried out on optional 6 clones among the positive clonesfound in the item (2).

Among the master plates cryo-preserved in the item (2), 96 well platescontaining the 6 positive clones were selected and incubated at 37° C.for 10 minutes in 5% CO₂. After the incubation, cells were collectedfrom a well corresponding to each positive clone and inoculated into awell flat bottom plate for adhesion cell use (manufactured by Greiner).After culturing them for 1 week using IMDM medium (manufactured by LifeTechnologies) supplemented with 10% fetal bovine dialyzed serum(manufactured by Life Technologies), the cells were inoculated into a 6well flat bottom plate for adhesion cell use (manufactured by Greiner).Genome DNAs were prepared from each clone in the plate in accordancewith a known method [Nucleic Acids Research, 3, 2303 (1976)] anddissolved overnight in 150 μl of a TE-RNase buffer (pH 8.0) (10 mmol/lTris-HCl, 1 mmol/l EDTA, 200 μg/ml RNase A).

In 120 μl of NEBuffer for BamHI (manufactured by New England Biolabs),12 μg of the obtained genome DNA was dissolved, and 20 unites of arestriction enzyme BamHI (manufactured by New England Biolabs) weremixed, followed by digestion reaction at 37° C. overnight. A DNAfragment was recovered from the reaction solution by ethanolprecipitation, dissolved in 20 μl of TE buffer (pH 8.0) (10 mmol/lTris-HCl, 1 mmol/l EDTA) and then subjected to 0.4% (w/v) agarose gelelectrophoresis. After the electrophoresis, the genome DNA wastransferred onto a nylon membrane in accordance with a known method[Proc. Natl. Acad. Sci. USA, 76, 3683 (1979)]. After completion of thetransfer, the nylon membrane was heated at 80° C. for 2 hours.

On the other hand, a probe used in the Southern blotting was prepared asfollows. First, primers (SEQ ID NOs:30 and 31) which bind to a sequenceoutside the targeting vector homologous region among the FUT8 genomeregion obtained in Example 12 were designed. Next, polymerase chainreaction (PCR) was carried out using a DNA polymerase ExTaq(manufactured by Takara Shuzo), by preparing 20 μl of a reactionsolution [ExTaq buffer (manufactured by Takara Shuzo), 0.2 mmol/l dNTPsand 0.5 μmol/l gene-specific primers (SEQ ID NOs:30 and 31)] containing4.0 ng of the plasmid pFUT8fgE2-2 obtained in Example 12(2). The PCR wascarried out by heating at 94° C. for 1 minute and subsequent 25 cyclesof heating at 94° C. for 30 seconds, 55° C. for 30 seconds and 74° C.for 1 minute as one cycle. After the PCR, the reaction solution wassubjected to 1.75% (w/v) agarose gel electrophoresis to purify a probeDNA fragment of about 230 bp. A 5 μl portion of the obtained probe DNAsolution was radioisotope-labeled using 1.75 MBq of [α-³²P]dCTP andMegaprime DNA Labeling System, dCTP (manufactured by Amersham PharmaciaBiotech).

The hybridization was carried out as follows. First, the nylon membranewas sealed in a roller bottle, and pre-hybridization was carried out at65° C. for 3 hours by adding 15 ml of a hybridization solution [5×SSPE,50×Denhaldt's solution, 0.5% (w/v) SDS, 100 μg/ml salmon sperm DNA].Next, the ³²P-labeled probe DNA was heat-denatured and put into thebottle and the nylon membrane was heated overnight at 65° C.

After the hybridization, the nylon membrane was soaked in 50 ml of2×SSC-0.1% (w/v) SDS and heated at 65° C. for 15 minutes. Afterrepeating the washing step twice, the membrane was soaked in 50 ml of0.2×SSC-0.1% (w/v) SDS and heated at 65° C. for 15 minutes. Afterwashing the nylon membrane, it was exposed to an X-ray film two nightsat −80° C. for development.

By the restriction enzyme BamHI treatment, a DNA fragment of about 19.0Kb was formed from a wild type FUT8 allele. Also, a DNA fragment ofabout 12.5 Kb was formed from an allele in which homologousrecombination with a targeting vector was generated. In addition, whenthe puromycin resistance gene (about 1.5 Kb) was deleted from the allelein which homologous recombination was generated, a DNA fragment of about11.0 Kb was formed by the same treatment.

By the method, such specific fragments of about 19.0 Kb and about 11.0Kb were found from the genome DNA of 5 clones among the 6 clones. Sincethe quantitative ratio of both fragments was 1:1, it was shown that thepuromycin resistance gene was deleted from the cell lines in which 1copy of the FUT8 genome region was disrupted. Hereinafter, one of theclone is called 1st.ΔFUT8 2-46-1. Also, results of the genome Southernblotting of the strain 1st.ΔFUT8 2-46-1, 1st.ΔFUT8 2-46 and 5-03 areshown in FIG. 41. Also, the strain 1st.ΔFUT8 2-46-1, as a name of2-46-1, has been deposited on Sep. 26, 2001, as FERM BP-7755 inInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology (AIST Tsukuba Central 6, 1-1, Higashi1-Chome Tsukuba-shi, Ibaraki-ken 305-8566 Japan)).

4. Purification of Antibody Produced by α-1,6-Fucosyltransferase (FUT8)Gene-Disrupted Cell Line

1st.ΔFUT8 2-46-1 obtained in the item 3 of this Example by disruptingone copy of the FUT8 allele was suspended in IMDM medium (manufacturedby Life Technologies) supplemented with 15 μg/ml of puromycin(manufactured by SIGMA) and 10% fetal bovine dialyzed serum(manufactured by Life Technologies) to give a density of 3×10⁵ cells/ml,and then 60 ml in total of the suspension was inoculated into two T182flasks for adhesion cell culture use (manufactured by Greiner). Afterculturing them for 3 days, the supernatant was discarded and changed toa total of 60 ml of EXCELL301 medium (manufactured by JRH Biosciences).

After culturing them at 37° C. for 7 days in a 5% CO₂ incubator, thenumber of intact cells was counted to confirm that their viability wasalmost the same (each 30% or less), and then each cell suspension wasrecovered. The cell suspension was centrifuged at 3,000 rpm at 4° C. for10 minutes, and the recovered supernatant was centrifuged at 10,000 rpmat 4° C. for 1 hour and then filtered using 150 ml capacity PES FilterUnit (manufactured by NALGENE) having a pore diameter of 0.22 μm.

Prosep-A HighCapacity (manufactured by bioPROCESSING) was packed in a0.8 cm diameter column to a thickness of 2 cm and washed with 10 ml of0.1 mol/l citrate buffer (pH 3.0) and 10 ml of 1 mol/l glycine/NaOH-0.15mol/l NaCl buffer (pH 8.6) in this order to effect equilibrate thecarrier. Next, 100 ml of each of the culture supernatant was passedthrough the column and washed with 50 ml of 1 mol/l glycine/NaOH-0.15mol/l NaCl buffer (pH 8.6). After washing it, the antibody absorbed toProsep-A was eluted using 2.5 ml of 0.1 mol/l citrate buffer (pH 3.0),the eluate was fractionated in 500 μl portions and each fraction wasneutralized by mixing with 100 μl of 2 mol/l Tris-HCl (pH 8.5). Twofractions containing the antibody at a high concentration (1.2 ml intotal) were selected by the BCA method [Anal. Biochem., 150, (1985)],combined and then dialyzed against 10 mol/l citrate-0.15 mol/l NaClbuffer (pH 6.0) at 4° C. for a whole day and night. After the dialysis,the antibody solution was recovered and subjected to sterile filtrationusing a 0.22 μm pore size Millex GV (manufactured by MILLIPORE).

5. In Vitro Cytotoxic Activity (ADCC Activity) of Antibody Produced byα-1,6-Fucosyltransferase (FUT8) Gene-Disrupted Cell Line

In order to evaluate in vitro cytotoxic activity of the anti-CCR4antibody purified in the item 4 of this Example, ADCC activity wasmeasured using the CCR4-positive cell line CCR4/EL-4 described inExample 8.

The CCR4/EL-4 cells subcultured in RPMI1640 medium (manufactured by LifeTechnologies) containing 10% fetal bovine serum (manufactured by LifeTechnologies) (hereinafter referred to as “RPMI1640-FBS(10)”) weresuspended in 500 μl of RPMI1640-FBS(10) at a density of 1×10⁶ cells, and3.7 MBq of Na₂ ⁵¹CrO₄ was added thereto, followed by culturing at 37° C.for 90 minutes to label the cells with a radioisotope. Aftercentrifugation at 1,200 rpm for 5 minutes, the supernatant was discardedand the target cells were suspended in 5 ml of RPMI1640-FBS(10). Thewashing step was repeated three times and then the cell suspension wasincubated for 30 minutes on ice for spontaneous dissociation of theradioactive substance. The washing step was again repeated twice andthen the cells were suspended in 5 ml of RPMI1640-FBS(10) to therebyprepare 2.0×10⁵ cells/ml of a target cell suspension.

Separately, 30 ml of venous blood was collected from a healthy person,gently mixed with 0.5 ml of heparin sodium (manufactured by ShimizuPharmaceutical) and then mixed with 30 ml of physiological saline(manufactured by Otsuka Pharmaceutical). After mixing them, 10 ml of themixture was gently overlaid on 4 ml of Lymphoprep (manufactured byNYCOMED PHARMA AS) and centrifuged at room temperature at 2,000 rpm for30 minutes. The separated mononuclear cell fractions were collected fromthe centrifugation tubes, combined and then suspended in 30 ml ofRPMI1640-FBS(10). After centrifugation at room temperature at 1,200 rpmfor 15 minutes, the supernatant was discarded and the cells weresuspended in 20 ml of RPMI1640-FBS(10). The washing step was repeatedtwice and then 2.5×10⁶ cells/ml of an effector cell suspension wasprepared using RPMI1640-FBS(10).

The target cell suspension was dispensed at 50 μl (1×10⁴ cells/well)into each well of a 96 well U-bottom plate (manufactured by Falcon).Subsequently, the effector cell suspension was dispensed at 100 μl(2.5×10⁵ cells/well) into each well to thereby adjust the ratio of theeffector cells to the target cells to 25:1. Next, usingRPMI1640-FBS(10), a series of dilution solution of 0.01 μg/ml, 0.1μg/ml, 1 μg/ml and 10 μg/ml was prepared from each of the anti-CCR4antibodies obtained in the item 5 of Example 13, and the dilutedsolutions were dispensed in 50 μl portions into the wells to give finalconcentrations of 0.0025 μg/ml, 0.025 μg/ml, 0.25 μg/ml and 2.5 μg/ml,respectively. After the reaction at 37° C. for 4 hours in 5% CO₂, theplate was centrifuged at 1,200 rpm for 5 minutes. Into a 12 mm diameterRIA tube (manufactured by IWAKI), 75 μl of the supernatant in each wellwas batched off and the amount of the dissociated ⁵¹Cr was measuredusing MINAX-γ auto-gamma counter 5550 (manufactured by PACKARD).

Also, the amount of the spontaneously dissociated ⁵¹Cr was calculated bycarrying out the same reaction in a reaction mixture in which 150 μl ofRPMI1640-FBS(10) was added instead of the effector cell suspension andantibody solution. The amount of the total dissociated ⁵¹Cr wascalculated by carrying out the same reaction in a reaction mixture inwhich 100 μl of 1 N hydrochloric acid and 50 μl of RPMI1640-FBS(10) wereadded instead of the effector cell suspension and antibody solution.Using these values, the ADCC activity was calculated based on equation(II).

FIG. 42 shows ADCC activity of each of the anti-CCR4 antibodies. Theantibody obtained from the strain 1st.ΔFUT8 2-46-1 in which 1 copy ofthe FUT8 allele was disrupted showed a significantly more potent ADCCactivity than the antibody produced by the strain 5-03 which is the CHOcell line before gene disruption. Also, changes in the antigen bindingactivity of these antibodies were not observed. Based on the results, itwas confirmed that the ADCC activity of produced antibodies can beimproved by disrupting the FUT8 allele in host cells.

Example 14 Preparation of Lectin-Resistant CHO/DG44 Cell and Productionof Antibody Using the Cell (1) Preparation of Lectin-Resistant CHO/DG44

CHO/DG44 cells were grown until they reached a stage of just beforeconfluent, by culturing in a 75 cm² flask for adhesion culture(manufactured by Greiner) using IMDM-FBS(10) medium [IMDM mediumcomprising 10% of fetal bovine serum (FBS) and 1× concentration of HTsupplement (manufactured by GIBCO BRL)]. After washing the cells with 5ml of Dulbecco PBS (manufactured by Invitrogen), 1.5 ml of 0.05% trypsin(manufactured by Invitrogen) diluted with Dulbecco PBS was added theretoand incubated at 37° C. for 5 minutes for peel the cells from the flaskbottom. The peeled cells were recovered by a centrifugation operationgenerally used in cell culture and suspended in IMDM-FBS(10) medium togive a density of 1×10⁵ cells/ml, and then 0.1 μg/ml of an alkylatingagent N-methyl-N′-nitro-N-nitrosoguanidine (hereinafter referred to as“MNNG”, manufactured by Sigma) was added or not added thereto. Afterincubating them at 37° C. for 3 days in a CO₂ incubator (manufactured byTABAI), the culture supernatant was discarded, and the cells were againwashed, peeled and recovered by the same operations, suspended inIMDM-FBS(10) medium and then inoculated into an adhesion culture 96 wellplate (manufactured by IWAKI Glass) to give a density of 1,000cells/well. To each well, as the final concentration in medium, 1 mg/mlLens culinaris agglutinin (hereinafter referred to as “LCA”,manufactured by Vector), 1 mg/ml Aleuria aurantia agglutinin (Aleuriaaurantia lectin; hereinafter referred to as “AAL”, manufactured byVector) or 1 mg/ml kidney bean agglutinin (Phaseolus vulgarisleucoagglutinin; hereinafter referred to as “L-PHA”, manufactured byVector) was added. After culturing them at 37° C. for 2 weeks in a CO₂incubator, the appeared colonies were obtained as lectin-resistantCHO/DG44. Regarding the obtained lectin-resistant CHO/DG44, anLCA-resistant cell line was named CHO-LCA, an AAL-resistant cell linewas named CHO-AAL and an L-PHA-resistant cell line was named CHO-PHA.When the resistance of these cell lines to various kinds of lectin wasexamined, it was found that the CHO-LCA was also resistant to AAL andthe CHO-AAL was also resistant LCA. In addition, the CHO-LCA and CHO-AALalso showed a resistance to a lectin which recognizes a sugar chainstructure identical to the sugar chain structure recognized by LCA andAAL, namely a lectin which recognizes a sugar chain structure in which1-position of fucose is bound to 6-position of N-acetylglucosamineresidue in the reducing end through α-bond in the N-glycoside-linkedsugar chain. Specifically, it was found that the CHO-LCA and CHO-AAL canshow resistance and survive even in a medium supplemented with 1 mg/mlat a final concentration of a pea agglutinin (Pisum sativum agglutinin;hereinafter referred to as “PSA”, manufactured by Vector). In addition,even when the alkylating agent MNNG was not added, it was able to obtainlectin-resistant cell lines by increasing the number of cells to betreated. Hereinafter, these cell lines were used in analyses.

(2) Preparation of Anti-CCR4 Human Chimeric Antibody-Producing Cell

An anti-CCR4 human chimeric antibody expression plasmid pKANTEX2160 wasintroduced into the three lectin-resistant cell lines obtained in the(1) by the method described in Example 8, and gene amplification by adrug MTX was carried out to prepare an anti-CCR4 human chimericantibody-producing cell line. By measuring an amount of antibodyexpression by the ELISA described in Example 8-2, antibody-expressingtransformants were obtained from each of the CHO-LCA, CHO-AAL andCHO-PHA. Regarding each of the obtained transformants, a transformantderived from CHO-LCA was named CHO/CCR4-LCA, a transformant derived fromCHO-AAL was named CHO/CCR4-AAL and a transformant derived from CHO-PHAwas named CHO/CCR4-PHA. Also, the CHO/CCR4-LCA, as a name of Nega-13,has been deposited on Sep. 26, 2001, as FERM BP-7756 in InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology (AIST Tsukuba Central 6, 1-1, Higashi 1-ChomeTsukuba-shi, Ibaraki-ken 305-8566 Japan)).

(3) Production of Potent ADCC Activity Antibody by Lectin-Resistant CHOCell

Using the three transformants obtained in the (2), purified antibodieswere obtained by the method described in Example 8-3. The antigenbinding activity of each of the purified anti-CCR4 human chimericantibodies was evaluated using the ELISA described in Example 8-2. Theantibodies produced by all transformants showed an antigen bindingactivity similar to that of the antibody produced by a recombinant cellline (strain 5-03) prepared in Example 8 using general CHO/DG44 cell asthe host. Using these purified antibodies, ADCC activity of each of thepurified anti-CCR4 human chimeric antibodies was evaluated in accordancewith the method described in Example 8-7. The results are shown in FIG.43. In comparison with the antibody produced by the strain 5-03, about100 folds-increased ADCC activity was observed in the antibodiesproduced by the CHO/CCR4-LCA and CHO/CCR4-AAL. On the other hand, nosignificant increase in the ADCC activity was observed in the antibodyproduced by the CHO/CCR4-PHA. Also, when ADCC activities of theantibodies produced by the CHO/CCR4-LCA and YB2/0 were compared inaccordance with the method described in Example 8-7, it was found thatthe antibody produced by the CHO/CCR4-LCA shows more potent ADCCactivity than the antibody produced by the strain 5-03, similar to thecase of the antibody KM2760-1 produced by the YB2/0 cell line preparedin Example 8-1 (FIG. 44).

(4) Sugar Chain Analysis of Antibodies Produced by Lectin-Resistant CHOCell

Sugar chains of the anti-CCR4 human chimeric antibodies purified in the(3) were analyzed. The solution of each of the purified antibodies wasexchanged to 10 mM KH₂PO₄ using Ultra Free 0.5-10K (manufactured byMillipore). The exchange was carried out in such a manner that theexchanging ratio became 80 folds or more. The concentration of theantibodies after the solution exchange was measured using UV-1600(manufactured by Shimadzu). The molar absorption coefficient wascalculated from the amino acid sequence of each antibody based on thefollowing equation (III) [Advances in Protein Chemistry, 12, 303(1962)], and the concentration was determined by defining the absorbanceat 280 nm as 1.38 mg/ml.

E _(1mol/l) =A×n1+B×n2+C×n3

E _(1mol/ml) =E _(1mol/l)/MW  (III)

-   -   E_(1mol/l): absorption coefficient at 280 nm (mg⁻¹ ml cm⁻¹)    -   E_(1mol/ml): molar absorption coefficient at 280 nm (M⁻¹ cm⁻¹)

-   A: molar absorption coefficient of tryptophan at 280 nm=5550 (M⁻¹    cm⁻¹)    -   B: molar absorption coefficient of tyrosine at 280 nm=1340 (M⁻¹        cm⁻¹)    -   C: molar absorption coefficient of cystine at 280 nm=200 (M⁻¹        cm⁻¹)    -   n1: the number of tryptophan per 1 antibody molecule    -   n2: the number of tyrosine per 1 antibody molecule    -   n3: the number of cystine per 1 antibody molecule    -   MW: molecular weight of antibody (g/mol)

Into Hydraclub S-204 test tube, 100 μg of each antibody was put anddried using a centrifugal evaporator. The dried sample in the test tubewas subjected to hydrazinolysis using Hydraclub manufactured by Hohnen.The sample was allowed to react with hydrazine at 110° C. for 1 hourusing a hydrazinolysis reagent manufactured by Hohnen hydrazinolysis[Method of Enzymology, 83, 263 (1982)]. After the reaction, hydrazinewas evaporated under a reduced pressure, and the reaction tube wasreturned to room temperature by allowing it to stand for 30 minutes.Next, 250 μl of an acetylation reagent manufactured by Hohnen and 25 μlof acetic anhydride were added thereto, followed by thoroughly stirredfor reaction at room temperature for 30 minutes. Then, 250 μl of theacetylation reagent and 25 μl of acetic anhydride were further addedthereto, followed by thoroughly stirring for reaction at roomtemperature for 1 hour. The sample was frozen at −80° C. in a freezerand freeze-dried for about 17 hours. Sugar chains were recovered fromthe freeze-dried sample using Cellulose Cartridge Glycan Preparation Kitmanufactured by Takara Syuzo Co., Ltd. The sample sugar chain solutionwas dried using a centrifugal evaporator and then subjected tofluorescence labeling with 2-aminopyridine [J. Biochem., 95, 197(1984)]. The 2-aminopyridine solution was prepared by adding 760 μl ofHCl per 1 g of 2-aminopyridine (1×PA solution) and diluting the solution10 folds with reverse osmosis purified water (10-folds diluted PAsolution). The sodium cyanoborohydride solution was prepared by adding20 μl of 1×PA solution and 430 μl of reverse osmosis purified water per10 mg of sodium cyanoborohydride. To the sample, 67 μl of a 10folds-diluted PA solution was added, followed by reaction at 100° C. for15 minutes and spontaneously cooled, and 2 μl of sodium cyanoborohydridewas further added thereto, followed by reaction at 90° C. for 12 hoursfor fluorescence labeling of the sample sugar chains. Thefluorescence-labeled sugar chain group (PA-treated sugar chain group)was separated from excess reagent using Superdex Peptide HR 10/30 column(manufactured by Pharmacia). This step was carried out using 10 mMammonium bicarbonate as the eluent at a flow rate of 0.5 ml/min and at acolumn temperature of room temperature, and using a fluorescencedetector of 320 nm excitation wavelength and 400 nm fluorescencewavelength. The eluate was recovered to 30 minutes after addition of thesample and dried using a centrifugal evaporator to be used as purifiedPA-treated sugar chains. Next, reverse phase HPLC analysis of thepurified PA-treated sugar chains was carried out using CLC-ODS column(manufactured by Shimadzu, φ6.0 nm×159 nm). The step was carried out ata column temperature of 55° C. and at a flow rate of 1 ml/min and usinga fluorescence detector of 320 nm excitation wavelength and 400 nmfluorescence wavelength. The column was equilibrated with a 10 mM sodiumphosphate buffer (pH 3.8) and elution was carried out for 80 minutes bya 0.5% 1-butanol linear density gradient. Each of the PA-treated sugarchain was identified by post source decay analysis of each peak of theseparated PA-treated sugar chains using matrix-assisted laser ionizationtime of flight mass spectrometry (MALDI-TOF-MS analysis), comparison ofelution positions with standards of PA-treated sugar chain manufacturedby Takara Syuzo, and reverse phase HPLC analysis after digestion of eachPA-treated sugar chain using various enzymes (FIG. 45). Each of thesugar chain content was calculated from each of the peak area ofPA-treated sugar chain by reverse HPLC analysis. A PA-treated sugarchain whose reducing end is not N-acetylglucosamine was excluded fromthe peak area calculation, because it is an impurity or a by-productduring preparation of PA-treated sugar chain.

The analysis was carried out in the same manner as in Example 11(6)using a sodium phosphate buffer (pH 3.8) as buffer A and a sodiumphosphate buffer (pH 3.8)+0.5% 1-butanol as buffer B.

In FIG. 45, the ratio of the α-1,6-fucose-free sugar chain group wascalculated from the area occupied by the peaks (i) to (iv) among (i) to(viii), and the ratio of the α-1,6-fucose-bound sugar chain group fromthe area occupied by the peaks (v) to (viii) among (i) to (viii).

Results of the sugar chain structure analysis of the purified anti-CCR4human chimeric antibodies produced by lectin-resistant cell lines areshown in Table 6. The result shows the analysis of sugar chains of theanti-CCR4 human chimeric antibody produced by lectin-resistant celllines. The ratio of α-1,6-fucose-free sugar chains (%) calculated frompeak areas by analyzing by the method described in Example d(4) is shownin the table.

TABLE 6 α-1,6-Fucose-free complex Antibody producing cells double-chainsugar chain (%) Strain 5-03 9 Strain CHO/CCR4-LCA 48 Strain CHO/CCR4-AAL27 Strain CHO/CCR4-PHA 8

In comparison with the antibody produced by the strain 5-03, the ratioof the α-1,6-fucose-free sugar chains was increased from 9% to 48% inthe antibody produced by the CHO/CCR4-LCA when calculated from theanalyzed peak area. The ratio of α-1,6-fucose-free sugar chains wasincreased from 9% to 27% in the antibody produced by the CHO/CCR4-AAL.On the other hand, changes in the sugar chain pattern and ratio of theα-1,6-fucose-free sugar chains were hardly found in the PHA-resistantcell line when compared with the strain 5-03.

Example 15 Analysis of Lectin-Resistant CHO Cell Line 1. Analysis ofExpression Level of GMD Enzyme in an Anti-CCR4 Human ChimericAntibody-Producing Cell Line CHO/CCR4-LCA

The expression level of each of the genes of GMD (GDP-mannose4,6-dehydratase), GFPP (GDP-keto-6-deoxymannose 3,5-epimerase,4-reductase) and FX (GDP-beta-L-fucose pyrophosphorylase) known asfucose biosynthesis enzymes and FUT8 (α-1,6-fucosyltransferase) as afucose transferase, in the anti-CCR4 human chimeric antibody-producingcell line CHO/CCR4-LCA obtained in Example 14, was analyzed using RT-PCRmethod.

(1) Preparation of RNA from Various Cell Lines

Each of the CHO/DG44 cell, the anti-CCR4 human chimericantibody-producing cell line 5-03 obtained in Example 8-1(2) and theanti-CCR4 human chimeric antibody-producing cell line CHO/CCR4-LCAobtained in Example 14(2) was subcultured at 37° C. in a 5% CO₂incubator and then cultured for 4 days. After culturing them, total RNAwas prepared from 1×10⁷ cells of each cell line using RNeasy ProtectMini Kit (manufactured by QIAGEN) in accordance with the manufacture'sinstructions. Subsequently, single-stranded cDNA was synthesized from 5μg of each RNA in a 20 μl of a reaction solution using SUPER SCRIPTFirst-Strand Synthesis System for RT-PCR (manufactured by GIBCO BRL) inaccordance with the manufacture's instructions.

(2) Analysis of Expression Level of GMD Gene Using RT-PCR

In order to amplify GMD cDNA by PCR, a 24 mer synthetic DNA primerhaving the nucleotide sequence shown by SEQ ID NO:32 and a 26 mersynthetic DNA primer having the nucleotide sequence shown by SEQ IDNO:33 were prepared based on the CHO cell-derived GMD cDNA sequenceshown in Example 17-1.

Next, 20 μl of a reaction solution [1×Ex Taq buffer (manufactured byTakara Shuzo), 0.2 mM dNTPs, 0.5 unit of Ex Taq polymerase (manufacturedby Takara Shuzo) and 0.5 μM of the synthetic DNA primers of SEQ IDNOs:32 and 33] containing 0.5 μl of the single-stranded cDNA preparedfrom each cell line in the item (1) as the template was prepared, andPCR was carried out using DNA Thermal Cycler 480 (manufactured by PerkinElmer) by heating at 94° C. for 5 minutes and subsequent 30 cycles ofheating of 94° C. for 1 minute and 68° C. for 2 minutes as one cycle.After subjecting 10 μl of the PCR reaction solution to agaroseelectrophoresis, DNA fragments were stained using Cyber Green(manufactured by BMA) and then the amount of the DNA fragment of about350 bp was measured using Fluor Imager SI (manufactured by MolecularDynamics).

(3) Analysis of Expression Level of GFPP Gene Using RT-PCR

In order to amplify GFPP cDNA by PCR, a 27 mer synthetic DNA primerhaving the nucleotide sequence shown by SEQ ID NO:34 and a 23 mersynthetic DNA primer having the nucleotide sequence shown by SEQ IDNO:35 were prepared based on the CHO cell-derived GFPP cDNA sequenceobtained in Example 16-2.

Next, 20 μl of a reaction solution [1×Ex Taq buffer (manufactured byTakara Shuzo), 0.2 mM dNTPs, 0.5 unit of Ex Taq polymerase (manufacturedby Takara Shuzo) and 0.5 μM of the synthetic DNA primers of SEQ IDNOs:34 and 35] containing 0.5 μl of the single-stranded cDNA preparedfrom each cell line in the item (1) as the template was prepared, andPCR was carried out using DNA Thermal Cycler 480 (manufactured by PerkinElmer) by heating at 94° C. for 5 minutes and subsequent 24 cycles ofheating at 94° C. for 1 minute and 68° C. for 2 minutes as one cycle.After subjecting 10 μl of the PCR reaction solution to agaroseelectrophoresis, DNA fragments were stained using Cyber Green(manufactured by BMA) and then the amount of the DNA fragment of about600 bp was measured using Fluor Imager SI (manufactured by MolecularDynamics).

(4) Analysis of Expression Level of FX Gene Using RT-PCR

In order to amplify FX cDNA by PCR, a 28 mer synthetic DNA primer havingthe nucleotide sequence shown by SEQ ID NO:36 and a 28 mer synthetic DNAprimer having the nucleotide sequence shown by SEQ ID NO:37 wereprepared based on the CHO cell-derived FX cDNA sequence shown in Example16-1.

Next, 20 μl of a reaction solution [1×Ex Taq buffer (manufactured byTakara Shuzo), 0.2 mM dNTPs, 0.5 unit of Ex Taq polymerase (manufacturedby Takara Shuzo) and 0.5 μM of the synthetic DNA primers of SEQ ID NO:36and SEQ ID NO:37] containing 0.5 μl of the single-stranded cDNA preparedfrom each cell line in the item (1) as the template was prepared, andPCR was carried out using DNA Thermal Cycler 480 (manufactured by PerkinElmer) by heating at 94° C. for 5 minutes and subsequent 22 cycles ofheating at 94° C. for 1 minute and 68° C. for 2 minutes as one cycle.After subjecting 10 μl of the PCR reaction solution to agaroseelectrophoresis, DNA fragments were stained using Cyber Green(manufactured by BMA) and then the amount of the DNA fragment of about300 bp was measured using Fluor Imager SI (manufactured by MolecularDynamics).

(5) Analysis of Expression Level of FUT8 Gene Using RT-PCR

In order to amplify FUT8 cDNA by PCR, 20 μl of a reaction solution [1×ExTaq buffer (manufactured by Takara Shuzo), 0.2 mM dNTPs, 0.5 unit of ExTaq polymerase (manufactured by Takara Shuzo) and 0.5 μM of thesynthetic DNA primers of SEQ ID NOs:13 and 14] containing 0.5 μl of thesingle-stranded cDNA prepared from each cell line in the item (1) as thetemplate was prepared, and PCR was carried out using DNA Thermal Cycler480 (manufactured by Perkin Elmer) by heating at 94° C. for 5 minutesand subsequent 20 cycles of heating at 94° C. for 1 minute and 68° C.for 2 minutes as one cycle. After subjecting 10 μl of the PCR reactionsolution to agarose electrophoresis, DNA fragments were stained usingCyber Green (manufactured by BMA) and then amount of the DNA fragment ofabout 600 bp was measured using Fluor Imager SI (manufactured byMolecular Dynamics).

(6) Analysis of Expression Level of β-Actin Gene Using RT-PCR

In order to amplify β-actin cDNA by PCR, 20 μl of a reaction solution[1×Ex Taq buffer (manufactured by Takara Shuzo), 0.2 mM dNTPs, 0.5 unitof Ex Taq polymerase (manufactured by Takara Shuzo) and 0.5 μM of thesynthetic DNA primers of SEQ ID NOs:15 and 16] containing 0.5 μl of thesingle-stranded cDNA prepared from each cell line in the item (1) as thetemplate was prepared, and the reaction was carried out using DNAThermal Cycler 480 (manufactured by Perkin Elmer) by heating at 94° C.for 5 minutes and subsequent 14 cycles of heating at 94° C. for 1 minuteand 68° C. for 2 minutes as one cycle. After subjecting 10 μl of the PCRreaction solution to agarose electrophoresis, DNA fragments were stainedusing Cyber Green (manufactured by BMA) and then the amount of the DNAfragment of about 800 bp was measured using Fluor Imager SI(manufactured by Molecular Dynamics).

(7) Expression Levels of GMD, GFPP, FX and FUT8 Genes in Each Cell Line

The amount of the PCR-amplified fragment of each gene in the strain 5-03and the CHO/CCR4-LCA was calculated by dividing values of the amounts ofPCR-amplified fragments derived from GMD, GFPP, FX and FUT cDNA in eachcell line measured in the items (2) to (6) by the value of the amount ofPCR-amplified fragment derived from β-actin cDNA in each cell line, anddefining the amount of the PCR-amplified fragments in CHO/DG44 cellas 1. The results are shown in Table 7.

TABLE 7 GMD GEPP FX FUT8 Strain CHO/DG44 1 1 1 1 Strain 5-03 1.107 0.7931.093 0.901 Strain 5-03-derived LCA- 0.160 0.886 0.920 0.875 resistantcell CHO/CCR4-LCA

As shown in Table 7, the expression level of GMD gene in theCHO/CCR4-LCA was decreased to about 1/10 in comparison with other celllines. In this case, the test was independently carried out twice, andthe average value was used.

2. Analysis Using Anti-CCR4 Human Chimeric Antibody-ProducingCHO/CCR4-LCA in which GMD Gene was Forced to Express(1) Construction of CHO Cell-Derived GMD Gene Expression VectorpAGE249GMD

Based on the CHO cell-derived GMD cDNA sequence obtained in Example17-1, a 28 mer primer having the nucleotide sequence shown by SEQ IDNO:38 and a 29 mer primer having the nucleotide sequence shown by SEQ IDNO:39 were prepared. Next, 20 μl of a reaction solution [1×Ex Taq buffer(manufactured by Takara Shuzo), 0.2 mM dNTPs, 0.5 unit of Ex Taqpolymerase (manufactured by Takara Shuzo) and 0.5 μM of the syntheticDNA primers of SEQ ID NOs:38 and 39] containing 0.5 μl of the CHOcell-derived GMD single-stranded cDNA prepared in the item 1(1) of thisExample as the template was prepared, and PCR was carried out using DNAThermal Cycler 480 (manufactured by Perkin Elmer) by heating at 94° C.for 5 minutes and subsequently 8 cycles of heating at 94° C. for 1minute, 58° C. for 1 minute and 72° C. for 1 minute as one cycle, andthen 22 cycles of heating at 94° C. for 1 minute and 68° C. as onecycle. After completion of the reaction, the PCR reaction solution wasfractionated by agarose electrophoresis, and then a DNA fragment ofabout 600 bp was recovered using Gene Clean II Kit (manufactured by BIO101) in accordance with the manufacture's instructions. The recoveredDNA fragment was connected to pT7Blue® vector (manufactured by Novagen)using DNA Ligation Kit (manufactured by Takara Shuzo), and E. coli DH5α(manufactured by Toyobo) was transformed using the obtained recombinantplasmid DNA to obtain a plasmid mt-C (cf. FIG. 46).

Next, based on the CHO cell-derived GMD cDNA sequence obtained inExample 17-1, a 45 mer primer having the nucleotide sequence shown bySEQ ID NO:40 and a 31 mer primer having the nucleotide sequence shown bySEQ ID NO:41 were prepared. Next, 20 μl of a reaction solution [1×Ex Taqbuffer (manufactured by Takara Shuzo), 0.2 mM dNTPs, 0.5 unit of Ex Taqpolymerase (manufactured by Takara Shuzo) and 0.5 μM of the syntheticDNA primers of SEQ ID NOs:40 and 41] containing 0.5 μl of the CHOcell-derived GMD single-stranded cDNA prepared in the item 1(1) of thisExample as the template was prepared, and PCR was carried out using DNAThermal Cycler 480 (manufactured by Perkin Elmer) by heating at 94° C.for 5 minutes and subsequently 8 cycles of heating at 94° C. for 1minute, 57° C. for 1 minute and 72° C. for 1 minute as one cycle, andthen 22 cycles of heating at 94° C. for 1 minute and 68° C. for 2minutes as one cycle. After completion of the reaction, the PCR reactionsolution was fractionated by agarose electrophoresis, and then a DNAfragment of about 150 bp was recovered using Gene Clean II Kit(manufactured by BIO 101) in accordance with the manufacture'sinstructions. The recovered DNA fragment was connected to pT7Blue®vector (manufactured by Novagen) using DNA Ligation Kit (manufactured byTakara Shuzo), and E. coli DH5α (manufactured by Toyobo) was transformedusing the obtained recombinant plasmid DNA to obtain a plasmid ATG (cf.FIG. 47).

Next, 3 μg of the plasmid CHO-GMD prepared in Example 17-1 was allowedto react with a restriction enzyme SacI (manufactured by Takara Shuzo)at 37° C. for 16 hours, a DNA was recovered by carrying outphenol/chloroform extraction and ethanol precipitation and allowed toreact with a restriction enzyme EcoRI (manufactured by Takara Shuzo) at37° C. for 16 hours, a digest DNA was fractionated by agaroseelectrophoresis and then a DNA fragment of about 900 bp was recoveredusing Gene Clean II Kit (manufactured by BIO 101) in accordance with themanufacture's instructions. The plasmid mt-C (1.4 μg) was allowed toreact with a restriction enzyme SacI (manufactured by Takara Shuzo) at37° C. for 16 hours, DNA was recovered by carrying out phenol/chloroformextraction and ethanol precipitation and allowed to react with arestriction enzyme EcoRI (manufactured by Takara Shuzo) at 37° C. for 16hours, the digest was fractionated by agarose electrophoresis and then aDNA fragment of about 3.1 kbp was recovered using Gene Clean II Kit(manufactured by BIO 101) in accordance with the manufacture'sinstructions. The recovered DNA fragments were ligated using DNALigation Kit (manufactured by Takara Shuzo), and E. coli DH5α wastransformed using the obtained recombinant plasmid DNA to obtain aplasmid WT-N(−) (cf. FIG. 48).

Next, 2 μg of the plasmid WT-N(−) was allowed to react with arestriction enzyme BamHI (manufactured by Takara Shuzo) at 37° C. for 16hours, DNA was recovered by carrying out phenol/chloroform extractionand ethanol precipitation and allowed to react with a restriction enzymeEcoRI (manufactured by Takara Shuzo) at 37° C. for 16 hours, the digestwas fractionated by agarose electrophoresis and then a DNA fragment ofabout 1 kbp was recovered using Gene Clean II Kit (manufactured by BIO101) in accordance with the manufacture's instructions. The plasmidpBluescript SK(−) (3 μg; manufactured by Stratagene) was allowed toreact with a restriction enzyme BamHI (manufactured by Takara Shuzo) at37° C. for 16 hours, DNA was recovered by carrying out phenol/chloroformextraction and ethanol precipitation and allowed to react with arestriction enzyme EcoRI (manufactured by Takara Shuzo) at 37° C. for 16hours, the digest was fractionated by agarose electrophoresis and then aDNA fragment of about 3 kbp was recovered using Gene Clean II Kit(manufactured by BIO 101) in accordance with the manufacture'sinstructions. The recovered respective DNA fragments were ligated usingDNA Ligation Kit (manufactured by Takara Shuzo), and E. coli DH5α wastransformed using the obtained recombinant plasmid DNA to obtain aplasmid WT-N(−) in pBS (cf. FIG. 49).

Next, 2 μg of the plasmid WT-N(−) in pBS was allowed to react with arestriction enzyme HindIII (manufactured by Takara Shuzo) at 37° C. for16 hours, DNA was recovered by carrying out phenol/chloroform extractionand ethanol precipitation and allowed to react with a restriction enzymeEcoRI (manufactured by Takara Shuzo) at 37° C. for 16 hours, the digestwas fractionated by agarose electrophoresis and then a DNA fragment ofabout 4 kbp was recovered using Gene Clean II Kit (manufactured by BIO101) in accordance with the manufacture's instructions. A 2 μg portionof the plasmid ATG was allowed to react with a restriction enzymeHindIII (manufactured by Takara Shuzo) at 37° C. for 16 hours, DNA wasrecovered by carrying out phenol/chloroform extraction and ethanolprecipitation and allowed to react with a restriction enzyme EcoRI(manufactured by Takara Shuzo) at 37° C. for 16 hours, the digest wasfractionated by agarose electrophoresis and then a DNA fragment of about150 bp was recovered using Gene Clean II Kit (manufactured by BIO 101)in accordance with the manufacture's instructions. The recoveredrespective DNA fragments were ligated using DNA Ligation Kit(manufactured by Takara Shuzo), and E. coli DH5α was transformed usingthe obtained recombinant plasmid DNA to obtain a plasmid WT in pBS (cf.FIG. 50).

Next, 2 μg of the plasmid pAGE249 was allowed to react with restrictionenzymes HindIII and BamHI (both manufactured by Takara Shuzo) at 37° C.for 16 hours, the digest was fractionated by agarose electrophoresis andthen a DNA fragment of about 6.5 kbp was recovered using Gene Clean IIKit (manufactured by BIO 101) in accordance with the manufacture'sinstructions. The plasmid WT (2 μg) in pBS was allowed to react withrestriction enzymes HindIII and BamHI (both manufactured by TakaraShuzo) at 37° C. for hours, the digest was fractionated by agaroseelectrophoresis and then a DNA fragment of about 1.2 kbp was recoveredusing Gene Clean II Kit (manufactured by BIO 101) in accordance with themanufacture's instructions. The recovered respective DNA fragments wereligated using DNA Ligation Kit (manufactured by Takara Shuzo), and E.coli DH5α was transformed using the obtained recombinant plasmid DNA toobtain a plasmid pAGE249GMD (cf. FIG. 51).

(2) Stable Expression of GMD Gene in CHO/CCR4-LCA

The CHO cell-derived GMD gene expression vector pAGE249GMD (5 μg) madeinto linear form by digesting it with a restriction enzyme FspI(manufactured by NEW ENGLAND BIOLABS), which was introduced into 1.6×10⁶cells of CHO/CCR4-LCA by electroporation [Cytotechnology, 3, 133(1990)]. Then, the cells were suspended in 30 ml of IMDM-dFBS(10) medium[IMDM medium (manufactured by GIBCO BRL) supplemented with 10% of dFBS]containing 200 nM MTX (manufactured by SIGMA), and cultured using a 182cm² flask (manufactured by Greiner) at 37° C. for 24 hours in a 5% CO₂incubator. After culturing them, the medium was changed to IMDM-dFBS(10)medium containing 0.5 mg/ml hygromycin and 200 nM MTX (manufactured bySIGMA), followed by culturing for 19 days to obtain colonies ofhygromycin-resistant transformants.

In the same manner, the pAGE249 vector was introduced into theCHO/CCR4-LCA by the same method to obtain colonies ofhygromycin-resistant transformants.

(3) Culturing of GMD Gene-Expressed CHO/CCR4-LCA and Purification ofAntibody

Using IMDM-dFBS(10) medium comprising 200 nM MTX (manufactured by SIGMA)and 0.5 mg/ml hygromycin, the GMD-expressing transformant cells obtainedin the item (2) were cultured using a 182 cm² flask (manufactured byGreiner) at 37° C. in a 5% CO₂ incubator. Several days thereafter, whenthe cell density reached confluent, the culture supernatant wasdiscarded and the cells were washed with 25 ml of PBS buffer(manufactured by GIBCO BRL) and mixed with 35 ml of EXCELL301 medium(manufactured by JRH). After culturing them at 37° C. in a 5% CO₂incubator for 7 days, the culture supernatant was recovered. Ananti-CCR4 chimeric antibody was purified from the culture supernatantusing Prosep-A (manufactured by Millipore) in accordance with themanufacture's instructions.

In the same manner, the pAGE249 vector-introduced transformant cellswere cultured by the same method and then anti-CCR4 chimeric antibodywas recovered and purified from the culture supernatant.

(4) Measurement of Lectin Resistance in Transformed Cells

The GMD-expressing transformant cells obtained in the item (2) weresuspended in IMDM-dFBS(10) medium comprising 200 nM MTX (manufactured bySIGMA) and 0.5 mg/ml hygromycin to give a density of 6×10⁴ cells/ml, andthe suspension was dispensed in 50 μl/well portions into a 96 wellculture plate (manufactured by Iwaki Glass). Next, a medium prepared bysuspending at concentrations of 0 mg/ml, 0.4 mg/ml, 1.6 mg/ml or 4 mg/mlLCA (Lens culinaris agglutinin: manufactured by Vector Laboratories) inIMDM-dFBS(10) medium containing 200 nM MTX (manufactured by SIGMA) and0.5 mg/ml hygromycin was added to the plate at 50 μl/well, followed byculturing at 37° C. for 96 hours in a 5% CO₂ incubator. After culturingthem, WST-I (manufactured by Boehringer) was added at 10 μl/well andincubated at 37° C. for 30 minutes in a 5% CO₂ incubator to effect colordevelopment, and then the absorbance at 450 nm and 595 nm (hereinafterreferred to as “OD450” and “OD595”, respectively) was measured usingMicroplate Reader (manufactured by BIO-RAD). In the same manner, thepAGE249 vector-introduced transformant cells were measured by the samemethod. The test was carried out twice independently.

FIG. 52 shows the number of survived cells in each well by percentagewhen a value calculated by subtracting OD595 from OD450 measured in theabove is used as the survived number of each cell group and the numberof survived cells in each of the LCA-free wells is defined as 100%. Asshown in FIG. 52, decrease in the LCA-resistance was observed in theGMD-expressed CHO/CCR4-LCA, and the survival ratio was about 40% in thepresence of 0.2 mg/ml LCA and the survival ratio was about 20% in thepresence of 0.8 mg/ml LCA. On the other hand, in the pAGE249vector-introduced CHO/CCR4-LCA, the survival ratio was 100% in thepresence of 0.2 mg/ml LCA and the survival ratio was about 80% even inthe presence of 0.8 mg/ml LCA. Based on these results, it was suggestedthat expression level of GMD gene in the CHO/CCR4-LCA was decreased and,as a result, the resistance against LCA was obtained.

(5) In Vitro Cytotoxic Activity (ADCC Activity) of Anti-CCR4 ChimericAntibody Obtained from GMD-Expressed CHO/CCR4-LCA

In order to evaluate in vitro cytotoxic activity of the purifiedanti-CCR4 chimeric antibody obtained in the item (3), the ADCC activitywas measured in accordance with the following methods.

i) Preparation of Target Cell Suspension

A 3.7 MBq equivalent of a radioactive substance Na₂ ⁵¹CrO₄ was added to1×10⁶ cells of the CCR4-EL4 (cf. Example 8-7) cultured using a mediumprepared by adding 500 μg/ml G418 sulfate (manufactured by NakalaiTesque) to the RPMI1640-FBS(10) medium, followed by reaction at 37° C.for 90 minutes to thereby label the cells with a radioisotope. After thereaction, the cells were washed three times by suspension in theRPMI1640-FBS(10) medium and subsequent centrifugation, re-suspended inthe medium and then incubated at 4° C. for 30 minutes in ice forspontaneous dissociation of the radioactive substance. Aftercentrifugation, the cells were adjusted to 2.5×10⁵ cells/ml by adding 5ml of the RPMI1640-FBS(10) medium and used as a target cell suspension.

ii) Preparation of Effector Cell Suspension

From a healthy person, 50 ml of venous blood was collected and gentlymixed with 0.5 ml of heparin sodium (manufactured by TakedaPharmaceutical). Using Lymphoprep (manufactured by Nycomed Pharma AS),the mixture was centrifuged in accordance with the manufacture'sinstructions to separate a mononuclear cell layer. The cells were washedthree times by centrifuging using the RPMI1640-FBS(10) medium and thenresuspended in the medium to give a density of 2×10⁶ cells/ml and usedas a effector cell suspension.

iii) Measurement of ADCC Activity

The target cell suspension prepared in the 1) was dispensed at 50 μl(1×10⁴ cells/well) into each well of a 96 well U-bottom plate(manufactured by Falcon). Next, 100 μl of the effector cell suspensionprepared in the 2) was added thereto (2×10⁵ cells/well, ratio of theeffector cells to the target cells was 25:1). Each of various anti-CCR4chimeric antibodies (the anti-CCR4 chimeric antibody purified in theitem (3), and KM2760-1 and KM3060) was further added thereto to give afinal concentration of 0.0025 to 2.5 μg/ml, followed by reaction at 37°C. for 4 hours. After the reaction, the plate was centrifuged and theamount of ⁵¹Cr in the supernatant was measured using a γ-counter. Theamount of the spontaneously dissociated ⁵¹Cr was calculated by carryingout the same procedure using the medium alone instead of the effectorcell suspension and antibody solution, and measuring the amount of ⁵¹Crin the supernatant. The amount of the total dissociated ⁵¹Cr wascalculated by carrying out the same procedure using the medium aloneinstead of the antibody solution and adding 1 N hydrochloric acidinstead of the effector cell suspension and measuring the amount of ⁵¹Crin the supernatant. The ADCC activity was calculated based on theformula (II).

Results of the measurement of ADCC activity are shown in FIG. 53. Asshown in FIG. 53, ADCC activity of the purified anti-CCR4 chimericantibody obtained from the GMD-expressed CHO/CCR4-LCA was decreased to asimilar degree to that of the KM3060 obtained in Example 8. On the otherhand, ADCC activity of the purified anti-CCR4 chimeric antibody obtainedfrom the pAGE249 vector-introduced CHO/CCR4-LCA showed a similar degreeof ADCC activity to that of the purified anti-CCR4 chimeric antibodyobtained from the CHO/CCR4-LCA. Based on the results, it was suggestedthat expression level of GMD gene in the CHO/CCR4-LCA is decreased and,as a result, an antibody having potent ADCC activity can be produced

(6) Sugar Chain Analysis of Anti-CCR4 Chimeric Antibody Derived fromGMD-Expressed CHO/CCR4-LCA

Sugar chains binding to the purified anti-CCR4 chimeric antibodyobtained in the item (3) were analyzed in accordance with the methodshown in Example 14(4), with the analyzed results shown in FIG. 55. Incomparison with the purified anti-CCR4 chimeric antibody prepared fromCHO/CCR4-LCA in Example 14, the ratio of sugar chain having noα-1,6-fucose in the purified anti-CCR4 chimeric antibody derived fromGMD-expressed CHO/CCR4-LCA was decreased to 9% when calculated from thepeak area. Thus, it was shown that the ratio of sugar chain having noα-1,6-fucose in the antibody produced by the cell is decreased tosimilar level of the antibody produced by the strain 5-03, by expressingGMD gene in the CHO/CCR4-LCA

Example 16 Preparation of Various Genes Encoding Enzymes Relating to TheSugar Chain Synthesis in CHO Cell

1. Determination of CHO Cell-Derived FX cDNA Sequence(1) Extraction of Total RNA from CHO/DG44 Cell

CHO/DG44 cells were suspended in IMDM medium containing 10% fetal bovineserum (manufactured by Life Technologies) and 1× concentration HTsupplement (manufactured by Life Technologies), and 15 ml of thesuspension was inoculated into a T75 flask for adhesion cell culture use(manufactured by Greiner) to give a density of 2×10⁵ cells/ml. On thesecond day after culturing them at 37° C. in a 5% CO₂ incubator, 1×10⁷of the cells were recovered and total RNA was extracted therefrom usingRNAeasy (manufactured by QIAGEN) in accordance with the manufacture'sinstructions.

(2) Preparation of Total Single-Stranded cDNA from CHO/DG44 Cell

The total RNA prepared in the (1) was dissolved in 45 μl of sterilewater, and 1 μl of RQ1 RNase-Free DNase (manufactured by Promega), 5 μlof the attached 10×DNase buffer and 0.5 μl of RNasin RibonucleaseInhibitor (manufactured by Promega) were added thereto, followed byreaction at 37° C. for 30 minutes to degrade genome DNA contaminated inthe sample. After the reaction, the total RNA was purified again usingRNAeasy (manufactured by QIAGEN) and dissolved in 50 μl of sterilewater.

In a 20 μl of reaction mixture using oligo(dT) as a primer,single-stranded cDNA was synthesized from 3 μg of the obtained total RNAsamples by carrying out reverse transcription reaction usingSUPERSCRIPT™ Preamplification System for First Strand cDNA Synthesis(manufactured by Life Technologies) in accordance with the manufacture'sinstructions. A 50 folds-diluted aqueous solution of the reactionsolution was used in the cloning of GFPP and FX. This was stored at −80°C. until use.

(3) Preparation of cDNA Partial Fragment of Chinese Hamster-Derived FX

FX cDNA partial fragment derived from Chinese hamster was prepared bythe following procedure.

First, primers (shown in SEQ ID NOs:42 and 43) specific for commonnucleotide sequences registered at a public data base, namely a human FXcDNA (Genebank Accession No. U58766) and a mouse cDNA (GenebankAccession No. M30127), were designed.

Next, 25 μl of a reaction solution [ExTaq buffer (manufactured by TakaraShuzo), 0.2 mmol/l dNTPs and 0.5 μmol/l gene-specific primers (SEQ IDNOs:42 and 43)] containing 1 μl of the CHO/DG44-derived single-strandedcDNA prepared in the item (2) was prepared, and polymerase chainreaction (PCR) was carried out using a DNA polymerase ExTaq(manufactured by Takara Shuzo). The PCR was carried out by heating at94° C. for 5 minutes, subsequent 30 cycles of heating at 94° C. for 1minute, 58° C. for 2 minutes and 72° C. for 3 minutes as one cycle, andfinal heating at 72° C. for 10 minutes.

After the PCR, the reaction solution was subjected to 2% agarose gelelectrophoresis, and a specific amplified fragment of 301 bp waspurified using QuiaexII Gel Extraction Kit (manufactured by Quiagen) andeluted with 20 μl of sterile water (hereinafter, the method was used forthe purification of DNA fragments from agarose gel). Into a plasmidpCR2.1, 4 μl of the amplified fragment was employed to insert inaccordance with the instructions attached to TOPO TA Cloning Kit(manufactured by Invitrogen), and E. coli DH5α was transformed with thereaction solution by the method of Cohen et al. [Proc. Natl. Acad. Sci.USA, 69, 2110 (1972)] (hereinafter, the method was used for thetransformation of E. coli). Plasmid DNA was isolated in accordance witha known method [Nucleic Acids Research, 7, 1513 (1979)] (hereinafter,the method was used for the isolation of plasmid) from the obtainedseveral kanamycin-resistant colonies to obtain 2 clones into which FXcDNA partial fragments were respectively inserted. They are referred toas pCRFX clone 8 and pCRFX clone 12.

The nucleotide sequence of the cDNA inserted into each of the FX clone 8and FX clone 12 was determined using DNA Sequencer 377 (manufactured byParkin Elmer) and BigDye Terminator Cycle Sequencing FS Ready Reactionkit (manufactured by Parkin Elmer) in accordance with the method of themanufacture's instructions. It was confirmed that each of the insertedcDNA whose sequence was determined encodes open reading frame (ORF)partial sequence of the Chinese hamster FX.

(4) Synthesis of Single-Stranded cDNA for RACE

Single-stranded cDNA samples for 5′ and 3′ RACE were prepared from theCHO/DG44 total RNA extracted in the item (1) using SMART™ RACE cDNAAmplification Kit (manufactured by CLONTECH) in accordance with themanufacture's instructions. In the case, PowerScript™ ReverseTranscriptase (manufactured by CLONTECH) was used as the reversetranscriptase. Each single-stranded cDNA after the preparation wasdiluted 10 folds with the Tricin-EDTA buffer attached to the kit andused as the template of PCR.

(5) Determination of Chinese Hamster-Derived FX Full Length cDNA by RACEMethod

Based on the FX partial sequence derived from Chinese hamster determinedin the item (3), primers FXGSP1-(SEQ ID NO:44) and FXGSP1-2 (SEQ IDNO:45) for the Chinese hamster FX-specific 5′ RACE and primers FXGSP2-1(SEQ ID NO:46) and FXGSP2-2 (SEQ ID NO:47) for the Chinese hamsterFX-specific 3′ RACE were designed.

Next, polymerase chain reaction (PCR) was carried out using Advantage2PCR Kit (manufactured by CLONTECH), by preparing 50 μl of a reactionsolution [Advantage2 PCR buffer (manufactured by CLONTECH), 0.2 mMdNTPs, 0.2 mmol/l Chinese hamster FX-specific primers for RACE and 1×concentration of common primers (manufactured by CLONTECH)] containing 1μl of the CHO/DG44-derived single-stranded cDNA for RACE prepared in theitem (4).

The PCR was carried out by repeating 20 cycles of heating at 94° C. for5 seconds, 68° C. for 10 seconds and 72° C. for 2 minutes as one cycle.

After completion of the reaction, 1 μl of the reaction solution wasdiluted 50-folds with the Tricin-EDTA buffer, and 1 μl of the dilutedsolution was used as a template. The reaction solution was againprepared and the PCR was carried out under the same conditions. Thetemplates, the combination of primers used in the first and second PCRsand the length of amplified DNA fragments by the PCRs are shown in Table8.

TABLE 8 Combination of primers used in Chinese hamster FX cDNA  RACE PCR and the size of PCR products FX-specific PCR-amplified 5′ RACEprimers Common primers product size First FXGSP1-1UPM (Universal primer mix)  300 bp Second FXGSP1-2NUP (Nested Universal primer) FX-specific PCR-amplified 3′ RACE primersCommon primers product size First FXGSP2-1 UPM (Universal primer mix)1,100 bp Second FXGSP2-2 NUP (Nested Universal primer)

After the PCR, the reaction solution was subjected to 1% agarose gelelectrophoresis, and the specific amplified fragment of interest waspurified using QiaexII Gel Extraction Kit (manufactured by Qiagen) andeluted with 20 μl of sterile water. Into a plasmid pCR2.1, 4 μl of theamplified fragment was inserted, and E. coli DH5α was transformed usingthe reaction solution in accordance with the instructions attached toTOPO TA Cloning Kit (manufactured by Invitrogen).

Plasmid DNAs were isolated from the appeared several kanamycin-resistantcolonies to obtain 5 cDNA clones containing Chinese hamster FX 5′region. They are referred to as FX5′ clone 25, FX5′ clone 26, FX5′ clone27, FX5′ clone 28, FX5′ clone 31 and FX5′ clone 32.

In the same manner, 5 cDNA clones containing Chinese hamster FX 3′region were obtained. These FX3′clones are referred to as FX3′ clone 1,FX3′ clone 3, FX3′ clone 6, FX3′ clone 8 and FX3′ clone 9.

The nucleotide sequence of the cDNA moiety of each of the clonesobtained by the 5′ and 3′ RACE was determined using DNA Sequencer 377(manufactured by Parkin Elmer) in accordance with the method describedin the manufacture's instructions. By comparing the cDNA nucleotidesequences determined by the method, reading errors of nucleotide basesdue to PCR were excluded and the full length nucleotide sequence ofChinese hamster FX cDNA was determined. The determined sequence is shownin SEQ ID NO:48.

2. Determination of CHO Cell-Derived GFPP cDNA Sequence(1) Preparation of GFPP cDNA Partial Fragment Derived from ChineseHamster

GFPP cDNA partial fragment derived from Chinese hamster was prepared bythe following procedure.

First, nucleotide sequences of a human GFPP cDNA (Genebank Accession No.number AF017445), mouse EST sequences having high homology with thesequence (Genebank Accession Nos. AI467195, AA422658, BE304325 andAI466474) and rat EST sequences (Genebank Accession Nos. BF546372,AI058400 and AW144783), registered at public data bases, were compared,and primers GFPP FW9 and GFPP RV9 (SEQ ID NOs:49 and 50) specific forrat GFPP were designed on a highly preserved region among these threespecies.

Next, polymerase chain reaction (PCR) was carried out using a DNApolymerase ExTaq (manufactured by Takara Shuzo), by preparing 25 μl of areaction solution [ExTaq buffer (manufactured by Takara Shuzo), 0.2 mMdNTPs and 0.5 μmol/l GFPP-specific primers GFPP FW9 and GFPP RV9 (SEQ IDNOs:49 and 50)] containing 1 μl of the CHO/DG44-derived single-strandedcDNA prepared in the item 1(2). The PCR was carried out by heating at94° C. for 5 minutes, subsequent 30 cycles of heating at 94° C. for 1minute, 58° C. for 2 minutes and 72° C. for 3 minutes as one cycle, andfinal heating at 72° C. for 10 minutes.

After the PCR, the reaction solution was subjected to 2% agarose gelelectrophoresis, and a specific amplified fragment of 1.4 Kbp waspurified using QuiaexII Gel Extraction Kit (manufactured by Quiagen) andeluted with 20 μl of sterile water. Into a plasmid pCR2.1, 4 μl of theamplified fragment was employed to insert in accordance with theinstructions attached to TOPO TA Cloning Kit (manufactured byInvitrogen), and E. coli DH5α was transformed using the reactionsolution.

Plasmid DNAs were isolated from the appeared several kanamycin-resistantcolonies to obtain 3 clones into which GFPP cDNA partial fragments wererespectively integrated. They are referred to as GFPP clone 8, GFPPclone 11 and GFPP clone 12.

The nucleotide sequence of the cDNA inserted into each of the GFPP clone8, GFPP clone 11 and GFPP clone 12 was determined using DNA Sequencer377 (manufactured by Parkin Elmer) and BigDye Terminator CycleSequencing FS Ready Reaction kit (manufactured by Parkin Elmer) inaccordance with the method described in the manufacture's instructions.It was confirmed that the inserted cDNA whose sequence was determinedencodes open reading frame (ORF) partial sequence of the Chinese hamsterGFPP.

(2) Determination of Chinese Hamster GFPP Full Length cDNA by RACEMethod

Based on the Chinese hamster FX partial sequence determined in the item2(1), primers GFPP GSP1-1 (SEQ ID NO:52) and GFPP GSP1-2 (SEQ ID NO:53)for the Chinese hamster FX-specific 5′ RACE and primers GFPP GSP2-1 (SEQID NO:54) and GFPP GSP2-2 (SEQ ID NO:55) for the Chinese hamsterGFPP-specific 3′ RACE were designed.

Next, polymerase chain reaction (PCR) was carried out using Advantage2PCR Kit (manufactured by CLONTECH), by preparing 50 μl of a reactionsolution [Advantage2 PCR buffer (manufactured by CLONTECH), 0.2 mMdNTPs, 0.2 mmol/l Chinese hamster GFPP-specific primers for RACE and 1×concentration of common primers (manufactured by CLONTECH)] containing 1μl of the CHO/DG44-derived single-stranded cDNA for RACE prepared in theitem (4).

The PCR was carried out by repeating 20 cycles of heating at 94° C. for5 seconds, 68° C. for 10 seconds and 72° C. for 2 minutes as one cycle.

After completion of the reaction, 1 μl of the reaction solution wasdiluted 50-folds with the Tricin-EDTA buffer, and 1 μl of the dilutedsolution was used as a template. The reaction solution was againprepared and the PCR was carried out under the same conditions. Thetemplates, the combination of primers used in the first and second PCRsand the size of amplified DNA fragments by the PCRs are shown in Table9.

TABLE 9 Combination of primers used in Chinese hamster GFPP cDNA RACE PCR and the size of PCR products GFPP-specific PCR-amplified 5′ RACEprimers Common primers product size First GFPPGSP1-1UPM (Universal primer mix) 1,100 bp Second GFPPGSP1-2NUP(Nested Universal primer) GFPP-specific PCR-amplified 3′ RACE primersCommon primers product size First GFPPGSP2-1 UPM (Universal primer mix)1,400 bp Second GFPPGSP2-2 NUP (Nested Universal primer)

After the PCR, the reaction solution was subjected to 1% agarose gelelectrophoresis, and the specific amplified fragment of interest waspurified using QiaexII Gel Extraction Kit (manufactured by Qiagen) andeluted with 20 μl of sterile water. Into a plasmid pCR2.1, 4 μl of theamplified fragment was employed to insert and E. coli DH5α wastransformed with the reaction solution in accordance with theinstructions attached to TOPO TA Cloning Kit (manufactured byInvitrogen).

Plasmid DNAs were isolated from the obtained several kanamycin-resistantcolonies to obtain 4 cDNA clones containing Chinese hamster GFPP 5′region. They are referred to as GFPP5′ clone 1, GFPP5′ clone 2, GFPP5′clone 3 and GFPP5′ clone 4.

In the same manner, 5 cDNA clones containing Chinese hamster GFPP 3′region were obtained. They are referred to as GFPP3′ clone 10, GFPP3′clone 16 and GFPP3′ clone 20.

The nucleotide sequence of the cDNA of each of the clones obtained bythe 5′ and 3′ RACE was determined using DNA Sequencer 377 (manufacturedby Parkin Elmer) in accordance with the method described in themanufacture's instructions. By comparing the cDNA nucleotide sequencesafter the nucleotide sequence determination, reading errors of bases dueto PCR were excluded and the full length nucleotide sequence of Chinesehamster GFPP cDNA was determined. The determined sequence is shown inSEQ ID NO:51.

Example 17 Preparation of CHO Cell-Derived GMD Gene

1. Determination of CHO Cell-Derived GMD cDNA Sequence(1) Preparation of CHO Cell-Derived GMD Gene cDNA (Preparation ofPartial cDNA Excluding 5′- and 3′-Terminal Sequences)

Rodents-derived GMD cDNA was searched in a public data base (BLAST)using a human-derived GMD cDNA sequence (GenBank Accession No. AF042377)registered at GenBank as a query, and three kinds of mouse EST sequenceswere obtained (GenBank Accession Nos. BE986856, BF158988 and BE284785).By ligating these EST sequences, a deduced mouse GMD cDNA sequence wasdetermined.

On the base of the mouse-derived GMD cDNA sequence, a 28 mer primerhaving the sequence represented by SEQ ID NO:56, a 27 mer primer havingthe sequence represented by SEQ ID NO:57, a 25 mer primer having thesequence represented by SEQ ID NO:58, a 24 mer primer having thesequence represented by SEQ ID NO:59 and a 25 mer primer having thesequence represented by SEQ ID NO:60 were generated.

Next, in order to amplify the CHO cell-derived GMD cDNA, PCR was carriedout by the following method. A 20 μl portion of a reaction solution[1×Ex Taq buffer (manufactured by Takara Shuzo), 0.2 mM dNTPs, 0.5 unitof Ex Taq polymerase (manufactured by Takara Shuzo) and 0.5 μM of twosynthetic DNA primers] containing 0.5 μl of the CHO cell-derivedsingle-stranded cDNA prepared in Example 15-1(1) as the template wasprepared. In this case, combinations of SEQ ID NO:56 with SEQ ID NO:57,SEQ ID NO:58 with SEQ ID NO:57, SEQ ID NO:56 with SEQ ID NO:59 and SEQID NO:56 with SEQ ID NO:60 were used as the synthetic DNA primers. Thereaction was carried out using DNA Thermal Cycler 480 (manufactured byPerkin Elmer) by heating at 94° C. for 5 minutes and subsequent 30cycles of heating at 94° C. for 1 minute and 68° C. for 2 minutes as onecycle.

The PCR reaction solution was fractionated by agarose electrophoresis tofind that a DNA fragment of about 1.2 kbp was amplified in the PCRproduct when synthetic DNA primers of SEQ ID NOs:56 and 57 were used, afragment of about 1.1 kbp was amplified in the PCR product whensynthetic DNA primers of SEQ ID NOs:57 and 59 were used, a fragment ofabout 350 bp was amplified in the PCR product when synthetic DNA primersof SEQ ID NOs:56 and 59 were used and a fragment of about 1 kbp wasamplified in the PCR product when synthetic DNA primers of SEQ ID NOs:56and 60 were used. The DNA fragments were recovered using Gene Clean IIKit (manufactured by BIO 101) in accordance with the manufacture'sinstructions. The recovered DNA fragments were ligated to a pT7Blue®vector (manufactured by Novagen) using DNA Ligation Kit (manufactured byTakara Shuzo), and E. coli DH (manufactured by Toyobo) was transformedusing the obtained recombinant plasmid DNA samples to thereby obtainplasmids 22-8 (having a DNA fragment of about 1.2 kbp amplified fromsynthetic DNA primers of SEQ ID NO:56 and SEQ ID NO:57), 23-3 (having aDNA fragment of about 1.1 kbp amplified from synthetic DNA primers ofSEQ ID NO:58 and SEQ ID NO:57), 31-5 (a DNA fragment of about 350 bpamplified from synthetic DNA primers of SEQ ID NO:56 and SEQ ID NO:59)and 34-2 (having a DNA fragment of about 1 kbp amplified from syntheticDNA primers of SEQ ID NO:56 and SEQ ID NO:60). The CHO cell-derived GMDcDNA sequence contained in these plasmids was determined in the usualway using a DNA sequencer ABI PRISM 377 (manufactured by Parkin Elmer)(since a sequence of 28 bases in downstream of the initiation codonmethionine in the 5′-terminal side and a sequence of 27 bases inupstream of the termination codon in the 3′-terminal side are originatedfrom synthetic oligo DNA sequences, they are mouse GMD cDNA sequences).

In addition, the following steps were carried out in order to prepare aplasmid in which the CHO cell-derived GMD cDNA fragments contained inthe plasmids 22-8 and 34-2 are combined. The plasmid 22-8 (1 μg) wasallowed to react with a restriction enzyme EcoRI (manufactured by TakaraShuzo) at 37° C. for 16 hours, the digest was subjected to agaroseelectrophoresis and then a DNA fragment of about 4 kbp was recoveredusing Gene Clean II Kit (manufactured by BIO 101) in accordance with themanufacture's instructions. The plasmid 34-2 (2 μg) was allowed to reactwith a restriction enzyme EcoRI at 37° C. for 16 hours, the digest wassubjected to agarose electrophoresis and then a DNA fragment of about150 bp was recovered using Gene Clean II Kit (manufactured by BIO 101)in accordance with the manufacture's instructions. The recovered DNAfragments were respectively subjected to terminal dephosphorylationusing Calf Intestine Alkaline Phosphatase (manufactured by Takara Shuzo)and then ligated using DNA Ligation Kit (manufactured by Takara Shuzo),and E. coli DH5α (manufactured by Toyobo) was transformed using theobtained recombinant plasmid DNA to obtain a plasmid CHO-GMD (cf. FIG.54).

(2) Determination of 5′-Terminal Sequence of CHO Cell-Derived GMD cDNA

A 24 mer primer having the nucleotide sequence represented by SEQ IDNO:61 was prepared from 5′-terminal side non-coding region nucleotidesequences of CHO cell-derived human and mouse GMD cDNA, and a 32 merprimer having the nucleotide sequence represented by SEQ ID NO:62 fromCHO cell-derived GMD cDNA sequence were prepared, and PCR was carriedout by the following method to amplify cDNA. Then, 20 μl of a reactionsolution [1×Ex Taq buffer (manufactured by Takara Shuzo), 0.2 mM dNTPs,0.5 unit of Ex Taq polymerase (manufactured by Takara Shuzo) and 0.5 μMof the synthetic DNA primers of SEQ ID NO:61 and SEQ ID NO:62]containing 0.5 μl of the single-stranded cDNA prepared in Example15-1(1) was prepared as the template, and the reaction was carried outtherein using DNA Thermal Cycler 480 (manufactured by Perkin Elmer) byheating at 94° C. for 5 minutes, subsequent 20 cycles of heating at 94°C. for 1 minute, 55° C. for 1 minute and 72° C. for 2 minutes as onecycle and further 18 cycles of heating at 94° C. for 1 minute and 68° C.for 2 minutes as one cycle. After fractionation of the PCR reactionsolution by agarose electrophoresis, a DNA fragment of about 300 bp wasrecovered using Gene Clean II Kit (manufactured by BIO 101) inaccordance with the manufacture's instructions. The recovered DNAfragment was ligated to a pT7Blue® vector (manufactured by Novagen)using DNA Ligation Kit (manufactured by Takara Shuzo), and E. coli DH5α(manufactured by Toyobo) was transformed using the obtained recombinantplasmid DNA samples to thereby obtain a plasmid 5′GMD. Using DNASequencer 377 (manufactured by Parkin Elmer), the nucleotide sequence of28 bases in downstream of the initiation methionine of CHO cell-derivedGMD cDNA contained in the plasmid was determined.

(3) Determination of 3′-Terminal Sequence of CHO Cell-Derived GMD cDNA

In order to obtain 3′-terminal cDNA sequence of CHO cell-derived GMD,RACE method was carried out by the following method. A single-strandedcDNA for 3′ RACE was prepared from the CHO cell-derived RNA obtained inExample 15-1(1) using SMART™ RACE cDNA Amplification Kit (manufacturedby CLONTECH) in accordance with the manufacture's instructions. In thecase, PowerScript™ Reverse Transcriptase (manufactured by CLONTECH) wasused as the reverse transcriptase. The single-stranded cDNA after thepreparation was diluted 10 folds with the Tricin-EDTA buffer attached tothe kit and used as the template of PCR.

Next, 20 μl of a reaction solution [ExTaq buffer (manufactured by TakaraShuzo), 0.2 mM dNTPs, 0.5 unit of EX Taq polymerase (manufactured byTakara Shuzo), 0.5 μM of the 25 mer synthetic DNA primer shown in SEQ IDNO:63 [generated on the base of the CHO cell-derived GMD cDNA sequencedetermined in the item (1)] and 1× concentration of Universal Primer Mix(attached to SMART™ RACE cDNA Amplification Kit; manufactured byCLONTECH] containing 1 μl of the cDNA for 3′ RACE as the template wasprepared, and PCR was carried out using DNA Thermal Cycler 480(manufactured by Perkin Elmer) by heating at 94° C. for 5 minutes andsubsequent 30 cycles of heating at 94° C. for 1 minute and 68° C. for 2minutes as one cycle.

After completion of the reaction, 1 μl of the PCR reaction solution wasdiluted 20 folds with Tricin-EDTA buffer (manufactured by CLONTECH).Then, 20 μl of a reaction solution [ExTaq buffer (manufactured by TakaraShuzo), 0.2 mM dNTPs, 0.5 unit of EX Taq polymerase (manufactured byTakara Shuzo), 0.5 μM of the 25 mer synthetic DNA primer shown in SEQ IDNO:64 [generated on the base of the CHO cell-derived GMD cDNA sequencedetermined in the item (1)] and 0.5 μM of Nested Universal Primer(attached to SMART™ RACE cDNA Amplification Kit; manufactured byCLONTECH) containing 1 μl of the 20 folds-diluted aqueous solution asthe template] was prepared, and the reaction was carried out using DNAThermal Cycler 480 (manufactured by Perkin Elmer) by heating at 94° C.for 5 minutes and subsequent 30 cycles at 94° C. for 1 minute and 68° C.for 2 minutes as one cycle.

After completion of the reaction, the PCR reaction solution wasfractionated by agarose electrophoresis and then a DNA fragment of about700 bp was recovered using Gene Clean II Kit (manufactured by BIO 101)in accordance with the manufacture's instructions. The recovered DNAfragment was ligated to a pT7Blue® vector (manufactured by Novagen)using DNA Ligation Kit (manufactured by Takara Shuzo), and E. coli DH5α(manufactured by Toyobo) was transformed using the obtained recombinantplasmid DNA, thereby obtaining a plasmid 3′GMD. Using DNA Sequencer 377(manufactured by Parkin Elmer), the nucleotide sequence of 27 bases inupstream of the termination codon of CHO cell-derived GMD cDNA containedin the plasmid was determined.

The full length cDNA sequence of the CHO-derived GMD gene determined bythe items (1), (2) and (3) and the corresponding amino acid sequence areshown in SEQ ID NOs:65 and 71, respectively.

2. Determination of Genomic Sequence Containing CHO/DG44-Derived CellGMD Gene

A 25 mer primer having the nucleotide sequence represented by SEQ IDNO:66 was prepared from the mouse GMD cDNA sequence determined inExample 17-1. Next, a CHO cell-derived genome DNA was obtained by thefollowing method. A CHO/DG44 cell-derived KC861 was suspended inIMDM-dFBS(10)-HT(1) medium [IMDM-dFBS(10) medium comprising 1×concentration of HT supplement (manufactured by Invitrogen)] to give adensity of 3×10⁵ cells/ml, and the suspension was dispensed at 2 ml/wellinto a 6 well flat bottom plate for adhesion cell use (manufactured byGreiner). After culturing them at 37° C. in a 5% CO₂ incubator until thecells became confluent on the plate, genome DNA was prepared from thecells on the plate by a known method [Nucleic Acids Research, 3, 2303(1976)] and dissolved overnight in 150 μl of TE-RNase buffer (pH 8.0)(10 mmol/l Tris-HCl, 1 mmol/l EDTA, 200 μg/ml RNase A).

A reaction solution (20 μl) [1×Ex Taq buffer (manufactured by TakaraShuzo), 0.2 mM dNTPs, 0.5 unit of EX Taq polymerase (manufactured byTakara Shuzo) and 0.5 μM of synthetic DNA primers of SEQ ID NO:59 andSEQ ID NO:66] containing 100 ng of the obtained CHO/DG44 cell-derivedgenome DNA was prepared, and PCR was carried out using DNA ThermalCycler 480 (manufactured by Perkin Elmer) by heating at 94° C. for 5minutes and subsequent 30 cycles of heating at 94° C. for 1 minute and68° C. for 2 minutes as one cycle. After completion of the reaction, thePCR reaction solution was fractionated by agarose electrophoresis andthen a DNA fragment of about 100 bp was recovered using Gene Clean IIKit (manufactured by BIO 101) in accordance with the manufacture'sinstructions. The recovered DNA fragment was ligated to a pT7Blue®vector (manufactured by Novagen) using DNA Ligation Kit (manufactured byTakara Shuzo), and E. coli DH5α (manufactured by Toyobo) was transformedusing the obtained recombinant plasmid DNA, thereby obtaining a plasmidex3. Using DNA Sequencer 377 (manufactured by Parkin Elmer), thenucleotide sequence of CHO cell-derived genome DNA contained in theplasmid was determined. The result is shown in SEQ ID NO:67.

Next, a 25 mer primer having the nucleotide sequence represented by SEQID NO:68 and a 25 mer primer having the nucleotide sequence representedby SEQ ID NO:69 were generated on the base of the CHO cell-derived GMDcDNA sequence determined in Example 17-1. Next, 20 μl of a reactionsolution [1×Ex Taq buffer (manufactured by Takara Shuzo), 0.2 mM dNTPs,0.5 unit of EX Taq polymerase (manufactured by Takara Shuzo) and 0.5 μMof the synthetic DNA primers of SEQ ID NO:68 and SEQ ID NO:69]containing 100 ng of the CHO/DG44-derived genome DNA was prepared, andPCR was carried out using DNA Thermal Cycler 480 (manufactured by PerkinElmer) by heating at 94° C. for 5 minutes and subsequent 30 cycles ofheating at 94° C. for 1 minute and 68° C. for 2 minutes as one cycle.

After completion of the reaction, the PCR reaction solution wasfractionated by agarose electrophoresis and then a DNA fragment of about200 bp was recovered using Gene Clean II Kit (manufactured by BIO 101)in accordance with the manufacture's instructions. The recovered DNAfragment was ligated to a pT7Blue® vector (manufactured by Novagen)using DNA Ligation Kit (manufactured by Takara Shuzo), and E. coli DH5α(manufactured by Toyobo) was transformed using the obtained recombinantplasmid DNA, thereby obtaining a plasmid ex4. Using DNA Sequencer 377(manufactured by Parkin Elmer), the nucleotide sequence of CHOcell-derived genome DNA contained in the plasmid was determined. Theresult is shown in SEQ ID NO:70.

Example 18 Sugar Chain Analysis of Conventionally Available Antibodies

Sugar chains binding to a conventionally available anti-HER2/neuantibody Herceptin (manufactured by GENENTECH and Roche) produced by CHOcell as the host cell was analyzed in accordance with the method ofExample 10(6) (FIG. 31). When calculated from each peak area of elutiondiagram, the content of α-1,6-fucose-free sugar chains of Herceptin was16%, and the content of α-1,6-fucose-bound sugar chains was 84%. Thesame analysis was carried out on other commercially availableantibodies, Rituxan (manufactured by GENENTECH, Roche and IDEC) andZenapax (manufactured by Roche and PDL), and the α-1,6-fucose-free sugarchain content of was less than that in Herceptin.

FIG. 31 is a graph showing elution pattern of PA-treated sugar chainsprepared from Herceptin, obtained by analyzing them by reverse phaseHPLC. The relative fluorescence intensity and the elution time areplotted as the ordinate and the abscissa, respectively. The reversephase HPLC analysis conditions, sugar chain structure analysis andcalculation of the ratio of sugar chain group containing no α-1,6-fucosesugar chain were carried out by the same methods of Example 11(6).

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skill in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof. All references cited hereinare incorporated in their entirety.

1-9. (canceled)
 10. A method for producing an antibody composition, saidmethod comprising culturing a cell in a cell culture medium to produceand accumulate an antibody composition in the culture; and recoveringthe antibody composition from the culture, wherein said cell is amammalian cell into which a gene encoding an antibody molecule isintroduced, and wherein said antibody composition comprises an antibodymolecule having complex N-glycoside-linked sugar chains bound to the Fcregion, wherein among the total complex N-glycoside-linked sugar chainsbound to the Fc region in the composition, the ratio of a sugar chain inwhich fucose is not bound to N-acetylglucosamine in the reducing end inthe sugar chain is 20% or more, wherein said mammalian cell is resistantto a lectin which recognizes a sugar chain in which the 1-position offucose is bound to the 6-position of N-acetylglucosamine in the reducingend through an α-bond in the complex N-glycoside-linked sugar chain,wherein said lectin resistance results from decreasing or deleting theactivity of an enzyme in said cell, said enzyme being selected from thegroup consisting of (a)-(d): (a) GDP-mannose 4,6-dehydratase; (b)GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase; (c)GDP-beta-L-fucose pyrophosphorylase; and (d) α1,6-fucosyltransferase,and wherein said antibody composition has greater antibody-dependentcellular cytotoxicity activity than an antibody composition produced byexpressing said gene in the parent mammalian cell that has not had theactivity of the enzyme selected from the group of (a)-(d) decreased ordeleted and that is not resistant to said lectin.
 11. The methodaccording to claim 10, wherein α1,6-fucosylation of the antibodycomposition is decreased or deleted by a technique selected from thegroup consisting of the following (a), (b), (c), (d) and (e): (a) a genedisruption technique targeting a gene encoding the enzyme; (b) atechnique for introducing a dominant negative mutant of a gene encodingthe enzyme; (c) a technique for introducing mutation into the enzyme;(d) a technique for inhibiting transcription or translation of a geneencoding the enzyme; (e) a technique for selecting a cell lineresistance to the lectin which recognizes a sugar chain in which the1-position of fucose is bound to the 6-position of N-acetylglucosaminein the reducing end through an α-bond in the complex N-glycoside-linkedsugar chain.
 12. The method according to claim 1, wherein the cell isselected from the group consisting of the following (a) to (i): (a) aCHO cell derived from a Chinese hamster ovary tissue; (b) a rat myelomacell line, YB2/3HL.P2.G11.16Ag.20 cell; (c) a mouse myeloma cell line,NSO cell; (d) a mouse myeloma cell line, SP2/0-Ag14 cell; (e) a BHK cellderived from a syrian hamster kidney tissue; (f) an antibody-producinghybridoma cell; (g) a human leukemia cell line Namalwa cell; (h) anon-human embryonic stem cell; (i) a non-human fertilized egg cell. 13.The method according to claim 1, wherein the lectin is selected from thegroup consisting of a Lens culinaris lectin, a pea lectin, a broad beanlectin and an Aleuria aurantia lectin.